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CHAPTER 7

OPTIMISATION AND DEVELOPMENT OF HERBAL

EXTRACT TREATED CURATIVE FABRICS FROM

METHANOLIC EXTRACT AND MICROENCAPSULATED

METHANOLIC OF COPPER ENRICHED

MEDICINAL HERBS

7.1 INTRODUCTION

This chapter deals about the development of herbal extract treated

curative fabrics. Methanolic extract and microencapsulated methanolic extract

of copper enriched medicinal herbs (Aerva lanata, Aloe barbadensis Mill,

Cumminum cyminum Linn, Tagetes erecta and Mentha piperita) were used

for this research work, and optimisation of the microcapsules coating process

parameter suitable for the development of curative textile products.

7.2 APPLICATION OF METHANOLIC EXTRACT AND

MICROCAPSULES ON COTTON FABRIC

The methanolic extract and microcapsules were applied on

bleached cotton fabric using pad – dry – cure method. Herbal extract was

applied on cotton fabric by two method namely with and without crosslinking

agent. The methanolic extracts from selected five medicinal herbs were

applied on the bleached cotton material with and without crosslinking agent

(each 5 replicates). The applications of microcapsules were carried out with

citric acid as crosslinking agent.

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The microcapsules produced from the methanolic extract of the five medicinal herbs were applied on the bleached cotton fabric. The various process parameters such as concentration of extract, concentration of cross linking agent and curing temperature were considered for the optimization process using Box and Behnken design and 15 samples were produced for each of the five selected herbs in various combinations as seen in Table 7.2.

7.2.1 Application of Methanolic Extract without Crosslinking Agents

The methanolic extract of active substance were diluted with water to material: liquor ratio of 1:10. The extracts were applied on bleached cotton fabric at 20% concentration by pad – dry cure method with 85% expression. The treated cotton fabric was then dried at 80oC for 20 minutes.

7.2.2 Application of Methanolic Extract with Crosslinking Agents

The methanolic extract of active substance were diluted with water to material: liquor ratio of 1:10. The extracts were applied on bleached cotton fabric along with crosslinking agent at 2.5% and extract concentration of 20% by pad – dry – cure method with 85% expression. The treated cotton fabric was then dried at 80oC for 20 minutes to remove the moisture and cured at 100oC.

7.2.3 Application of Microcapsules with Citric Acid as Crosslinking Agent

The microcapsules prepared from the methanolic extract of the five selected medicinal herbs and were applied to the bleached cotton fabric using citric acid as crosslinking agent. The process parameters were optimized using experimental design of Box and Behnken method with three independent variable and three levels in each variable. In this process there are three variable parameters, namely concentration of extract, concentration of cross linking agent and curing temperature. The bleached cotton fabrics

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were treated with crosslinking agent citric acid by according to the process condition variable in the Box and Behnken experimental plan as given in Table 7.1. In all the cases the material: liquor ratio was 1:10. The samples were produced by pad – dry – cure method.

7.3 OPTIMISATION OF PROCESS PARAMETERS

For the process optimization, the experimental design was done using Box and Behnken method with three independent variable and three level of each variable.

7.3.1 Process Optimization by Response Surface Methodology

The relationship between the set of controlled experimental factors and the obtained results was evaluated by response surface methodology. In this work, concentration of extract, cross linking agent and curing temperature were taken as significant variables and designated as X1, X2 and X3 respectively. Table 7.1 shows the lower, middle and higher levels of the variables that are designated as -1,0 and +1 respectively.

Table 7.1 Levels of variables taken for the trials

VariablesCoded Levels -1 0 -1

Concentration of extract (in % ) (X1) 15 20 25 Concentration of Cross linking agent (in %) (X2) 2.0 2.5 3.0

Curing temperature (oC) (X3) 90 100 110

The computation was carried out using multiple regression analysis

using the least squares method. In a system involving three significant

independent variables X1, X2, and X3, the mathematical relationship of the

response on these variables can be approximated by the given quadratic

(second degree) polynomial equation.

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Y=Co+C1X1+C2X2+C3X3+C12X1X2+C13X1X3+C23X2X3+C11X12+C22 X2

2+C33X32

(7.1)

where Y= Predicted yield, C0 = Constant,C1, C2 and C3 are Linear Coefficients, C12, C13 and C23 are Cross Product Coefficients,C11, C22 and C33 are Quadratic Coefficients.

A multiple regression analysis was used to obtain the coefficients.

Since relatively few experimental combinations of the variables were sufficient

to estimate complex response functions potentially, the Box-Behnken design

was chosen. To obtain the required 10 coefficients of the model, a total of

15 experiments were necessary.

Human pathogenic bacterial strains of Staphylococcus aureus and

Escherichia coli are most commonly used to assess antimicrobial activities

and the bacterial inhibition studies were carried out for these strains. The

Table 7.2 shows coating process optimized with process parameters.

7.3.2 Model Fitting and Statistical Analysis

The experimental data was analyzed using the statistical software,

Design Expert software version 8 for regression analysis to fit the equations

developed and also for the evaluation of the statistical significance of the

equations.

7.3.3 Validation of Optimization Model

Statistical significance of the equation was determined with

ANOVA feature of the Design Expert Software. Subsequently, the feasibility

and grid searches were performed to locate the composition of optimum

formulations. The contour plots were drawn using the software. The resultant

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experimental data of response properties were compared with that of the

predicted values and linear regression plots between the observed and predicted values of the response variables were drawn.

Table 7.2 Experimental plan for applied of microcapsules using five medicinal herbal extract using Box – Behnken experimental design

The microcapsules of methanolic extract from the five medicinal herbs Aerva lanata, Aloe barbadensis Mill, Cumminum cyminum Linn,Tagetes erecta and Mentha piperita were used for the experiment

Sample No Concentration

of extract (in %)

Concentration of cross linking

agent (in %)

Curing temperature

(oC)1 15 2.0 1002 25 2.0 1003 15 3.0 1004 25 3.0 1005 15 2.5 906 25 2.5 907 15 2.5 1108 25 2.5 1109 20 2.0 90

10 20 3.0 9011 20 2.0 11012 20 3.0 11013 20 2.5 10014 20 2.5 10015 20 2.5 100

The Table 7.2 shows the various combinations of selected copper

enriched medicinal herbs applied on the cotton fabric using pad – dry – cure

method. The various process parameters such as concentration of extract,

concentration of cross linking agent and curing temperature were considered

for the optimization process using Box and Behnken design and 15 samples

were produced for each selected medicinal herbs.

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7.4 RESULT AND DISCUSSION

As per standard testing procedure of qualitative analysis (SN195920)

and qualitative analysis AATCC 100, the assessment of antimicrobial activity

of methanolic extracts treated cotton samples with and without crosslinking

agent (5 samples) and microcapsules treated samples of the selected five

copper enriched medicinal herbs (15 samples each) were carried out.

7.4.1 Assessment of Methanolic Extract Treated Cotton Fabric

without Crosslinking Agent

The antimicrobial activity of methanolic extracts of copper

enriched medicinal herbal treated fabric without any crosslinking agent against

the control sample has been assessed by measuring the zone of inhibition using

agar diffusion method as described in SN195920 (Qualitative analysis) and

bacterial reduction % using challenge test (Quantitative analysis) AATCC 100.

Table 7.3 shows the antimicrobial activity of copper enriched herbs of Aerva

lanata, Aloe barbadensis Mill, Cumminum cyminum Linn, Tagetes erecta and

Mentha piperita showed maximum antimicrobial activity qualitatively and

quantitatively.

Table 7.3 Antimicrobial test result of methanolic extract treated cotton fabric without crosslinking agent

Samples

Qualitative analysis Quantitative analysis Zone of inhibition (mm) Bacterial reduction %

Staphylococcus aureus

Escherichia coli

Staphylococcus aureus

Escherichia coli

Aerva lanata 30 24 85.34 70.48Aloe barbadensis Mill 37 23 85.21 71.0

Cumminum cyminum Linn 26 23 81.98 69.56Tagetes erecta 34 25 86.45 69.12Mentha piperita 31 25 85.21 68.44

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Figure 7.1 Antimicrobial activity of methanolic extract treated cotton fabric without crosslinking agent against Staphylococcus aureus and Escherichia coli (a & b Aerva lanata, c & d Aloe barbadensis Mill, e & f Cumminum cyminumLinn, g & h Tagetes erecta, i & j Mentha piperita)

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The Figure 7.1 antimicrobial activity of methanolic extract treated

cotton fabric without crosslinking agent, based on the observation from the

figure and table, the methanolic extract (without crosslinking) treated cotton

fabric gave better antimicrobial activity against Staphylococcus aureus and

Escherichia coli. The zones of inhibition of methanolic extract treated fabrics

were 26-34 mm against Staphylococcus aureus and 23-25 mm against

Escherichia coli (Thilagavathi et al 2007; Mohanraj et al 2012).

7.4.2 Assessment of Methanolic Extract Treated Cotton Fabric with

Citric Acid as Crosslinking Agent

The Table 7.4 shows antimicrobial activity of methanolic extract

(Aerva lanata, Aloe barbadensis Mill, Cumminum cyminum Linn, Tagetes

erecta, and Mentha piperita) treated cotton fabric with citric acid as

crosslinking agent against the control sample has been assessed by measuring

the zone of inhibition using agar diffusion method as SN195920 (Qualitative

analysis) and bacterial reduction % using challenge test (Quantitative

analysis) AATCC 100 against Staphylococcus aureus and Escherichia coli.

Figure 7.2 Antimicrobial activity of methanolic extract treated cotton fabric with crosslinking agent (a & b herbs treated fabrics against Staphylococcus aureus) (c & d herbs treated fabrics against Escherichia coli)

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Table 7.4 Antimicrobial test result of methanolic extract treated cotton fabric with crosslinking agent of citric acid

Samples

Qualitative analysis Quantitative analysis Zone of inhibition (mm) Bacterial reduction %

Staphylococcus aureus

Escherichia coli

Staphylococcus aureus

Escherichia coli

Aerva lanata 38 27 89.48 76.56

Aloe barbadensis Mill 41 23 91.56 78.44

Cumminum cyminum Linn 40 23 90.72 73.48

Tagetes erecta 37 22 87.46 69.59

Mentha piperita 39 27 89.13 70.05

The agar diffusion test and challenge test results shows the better

maximum zone of inhibition and bacterial reduction % and it is given in

Table 7.4. Based on the observations from this study, the crosslinking also

influence to create bonding between fibre morphology and methanolic

extracts and give a better penetration of methanolic extract of medicinal herbs

into cotton fabric when compared to without crosslinking agent. (Thilagavathi

et al 2007).

7.4.3 Optimization of Process Parameters

The design matrixes of the variables in the actual units along with

their antimicrobial activity of microencapsulated of copper enriched

medicinal herbal (ie., Aerva lanata, Aloe barbadensis Mill, Cumminum

cyminum Linn, Tagetes erecta and Mentha piperita) treated cotton fabric

against with Staphylococcus aureus and Escherichia coli are given in

Table 7.5.

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Table 7.5 Antibacterial activity against Staphylococcus aureus and Escherichia coli corresponding to changes in experimental combinations

Factor Level combination

No

Concentrationof extract (%)

Concentration of cross linking

agent (%)

Curingtemperature

(oC)

Aerva lanata Aloe barbadensisMill

Cumminum cyminumLinn Tagetes erecta Mentha piperita

S. aureus E.coli S. aureus E.coli S. aureus E.coli S. aureus E.coli S. aureus E.coli1 15 2 100 20 22 39 25 23 21 24 21 22 20

2 25 2 100 19 21 35 22 26 22 27 24 25 23

3 15 3 100 19 20 43 30 21 19 22 19 20 19

4 25 3 100 20 21 32 20 25 21 26 22 24 20

5 15 2.5 90 19 19 43 29 21 20 22 21 20 19

6 25 2.5 90 22 19 44 28 23 21 24 23 22 22

7 15 2.5 110 24 23 40 26 23 19 24 20 22 19

8 25 2.5 110 24 20 42 28 25 20 26 20 24 20

9 20 2 90 22 19 36 23 21 21 22 21 20 22

10 20 3 90 20 19 31 19 21 19 22 20 20 21

11 20 2 110 20 19 42 28 29 20 28 20 27 21

12 20 3 110 20 19 35 22 21 19 22 19 20 20

13 20 2.5 100 20 21 42 26 31 26 30 25 30 24

14 20 2.5 100 19 23 37 23 29 26 29 26 28 23

15 20 2.5 100 23 24 38 25 31 25 31 26 30 23

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Based on the antimicrobial activity against Staphylococcus aureus

and Escherichia coli of microcapsules treated cotton fabric analyzed from the

measured responses by the Design-expert software, the fit summary output

indicated that the linear and quadratic polynomial models were significant for

the present system.

7.4.4 Model Building and Statistical Analysis of Microbial Protection

against Staphylococcus aureus and Escherichia coli

The response surface equations with significant factors were

obtained using Design-Expert V8 software. The final mathematical model in

terms of coded factors as determined by Design-expert software after

eliminating the insignificant factors are shown in the Table 7.6. In general, the

p values are used as a tool to verify the significance of each co-efficient,

which also indicates the microbial protection against Staphylococcus aureus

and Escherichia coli between each independent variable. The regression

coefficient (R2) represents the proportion that the model can explain for the

variation in the response. The high values of R2 (Table 7.6) for all the

microencapsulated copper enriched herbal plants (Aerva lanata, Aloe

barbadensis Mill, Cumminum cyminum Linn, Tagetes erecta and Mentha

piperita) shows the better antimicrobial activity against Staphylococcus

aureus and Escherichia coli were well correlated with all the chosen

variables.

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Table 7.6 Response surface regression equation for copper enriched medicinal herbs against Staphylococcus aureus and Escherichia coli

Sl. No

Herbs Regression Equations with

significant factors P Value R2 Value

1 Aerva lanata

Y1 = 23.66 + 0.75X1 - 0.625X2 + 0.6257X3 - 0.75 X2 X3 - 1.458X1

2 - 1.708X22 -

2.208X32

0.0028 0.969

Y2 = 23.33 + 0.625X1 - 0.5X2 + 0.125X3 - 0.25X1X2 - 0.75X2X3 - 1.666 X1

2 - 1.417X2

2 - 2.167 X32

0.0041 0.964

2

Aloe barbadensisMill

Y1 = 30.33333+1.375X1-1X2+1.125X3-1.25X2X3-2.91667X1

2-3.66667X22-

4.41667X32

0.0017 0.97515

Y2 = 25.66667+0.875X1-0.625X2-0.75X3-1.70833X1

2-2.70833X22-2.95833X3

2 0.0028 0.969383

3Cumminum

cyminum Linn

Y1 = 30.33333+1.375X1-1.375X2+1.5X3-2X2

X32-3.29167 X1

2-3.29167X22-4.04167X3

2 0.0074 0.954352

Y2 = 25.66667+0.625X1-0.75X2-0.375X3-2.3333 X1

2-2.58333X22-3.33333X3

2 0.0002 0.989207

4 Tagetes erecta

Y1 = 30+1.375X1-1.125X2+1.25X3-1.5X2X3-2.375X1

2-2.875X22-3.625X3

2 0.0037 0.965811

Y2 = 25.66667+1X1-0.75X2-0.75X3-1.58333X1

2-2.58333X22-3.08333X3

2 0.0070 0.955502

5 Mentha piperita

Y1 = 29.33333+1.375X1-1.25X2+1.375X3-1.75X2X3-3.16667X1

2-3.41667X22-

4.16667X32

0.0044 0.963131

Y2 = 23.33333+1X1-0.75X2-0.5X3-1.91667X1

2-0.91667X22-1.41667X3

2 0.0027 0.970034

Y1 & Y2-Significance level for microbial protection against Staphylococcus aureus (Y1) and Escherichia coli (Y2)

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7.4.5 Influence of Process Variables on Antimicrobial Activity

against Staphylococcus aureus

As per the experimental plan, 15 samples were treated with

microcapsules under optimized parameters, based on results, the influence of

process parameters on antimicrobial activity against Staphylococcus aureus

are discussed below.

7.4.5.1 Aerva lanata

The contour plots in Figure 7.3 shows the effect of process

parameters on microbial protection against Staphylococcus aureus with

microencapsulated Aerva lanata treated sample.

Figure 7.3 (a) shows the antimicrobial activity against extract

concentration with cross linking agent percentage. From the graph, it is clear

that the antimicrobial activity of the treated sample increases with the increase

in the extract concentration and the cross linking agent percentage up to

certain level and then decreases (Muralidhar 2001). At 21% extract

concentration and 2.4% cross linking agent concentration, the zone of

inhibition was maximum and it was found to be 23 mm. Figure 7.3 (b)

represents the contour plot, plotted against zone of inhibition versus extract

concentration with curing temperature. Here the increase in extract

concentration% and the curing temperature increases the antimicrobial activity

of the extract treated textile material up to certain level and then decreases as said

above and found to be maximum at 21% extract concentration and 1020C

curing temperature with the zone of inhibition of 23 mm.

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(a) (b)

(c)

Figure 7.3 Optimization of (a) extract concentration with cross linking agent (b) extract concentration with curing temperature (c) cross linking agent with curing Temperature on Staphylococcus aureus

Figure 7.3 (c) represents the activity against cross linking agent

percentage with curing temperature. The antimicrobial activity increases with

the increase in cross-linking agent concentration and curing temperature.

Better activity was observed with 2.5% cross linking agent and 1000C curing

temperature.

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7.4.5.2 Aloe barbadensis Mill

By analyzing response surface equation it can be shown that the

maximum antimicrobial activity of microencapsulated Aloe barbadensis Mill

treated cotton fabric were obtained at concentration of 21.19% with 2.44%

cross linking agent and curing temperature of the 100.03oC. Based on the

results from the contour plots, it was observed that the trend is similar to that

case of microencapsulated Aerva lanata treated sample. The ANOVA tables

are shown in Appendix 3.

7.4.5.3 Cumminum cyminum Linn

From response surface equation it can be shown that the maximum

antimicrobial activity of microencapsulated Cumminum cyminum Linn

treated sample were obtained at concentration of 22.96% with 2.42% cross

linking agent and curing temperature of 100.67oC. From the contour plots

were observed that the trend is similar to that case of microencapsulated

Aerva lanata treated sample. The ANOVA tables are shown in Appendix 3.

7.4.5.4 Tagetes erecta

Results from the response surface equation were observed that the

maximum antimicrobial activity in zone of inhibition in mm obtained from

the microencapsulated Tagetes erecta treated sample, at the level of

concentration in 21.47% with 2.42% cross linking agent and curing

temperature of 100.27oC. Based on the results from the contour plots, were

observed that the trend is similar to that case of microencapsulated Aerva

lanata treated sample. The ANOVA tables are shown in Appendix 3.

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7.4.5.5 Mentha piperita

By analyzing response surface equation it can be shown that the

maximum antimicrobial activity of microencapsulated Mentha piperita

treated sample were obtained at concentration of 21.28% with 2.36% cross

linking agent and curing temperature of 100.4oC. The contour plots shows the

influence of process parameters on antimicrobial activity against

Staphylococcus aureus of Mentha piperita treated sample, based on the

results from the contour plots, were observed that the trend is similar to that

case of microencapsulated Aerva lanata treated sample. The ANOVA tables

are shown in Appendix 3.

7.4.6 Influence of Process Variables on Antimicrobial Activity

against Escherichia coli

The contour plots in Figure 7.4 shows the effect of process

parameters on microbial protection against Escherichia coli with

microcapsules treated Aerva lanata treated sample. It was found that the

activity increases with the increase in extract concentration, cross liking agent

percentage and curing temperature up to certain point and then decreases as

mentioned above. The optimum conditions for obtaining the maximum

inhibition zone (23 mm) was 21% extract concentration, 2.4% cross linking

agent and 1000 C curing temperature. The shape of the contour plots (circular

or elliptical) indicates whether the mutual interactions between variables are

significant or not. A circular contour plot indicates that the interactions

between related variables are negligible. An elliptical contour plot indicates

that the interactions between related variables are significant (Annadurai &

Sheeja 1988, Kumar 2007).

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(a) (b)

(c)

Figure 7.4 Optimization of (a) Extract concentration with cross linking agent (b) Extract concentration with Curing temperature (c) Cross linking agent with Curing Temperature on Escherichia coli

By analyzing response surface equation and contour plots of

microencapsulated copper enriched herbs of Aerva lanata, Aloe barbadensis

Mill, Cumminum cyminum Linn, Tagetes erecta and Mentha piperita treated

samples, process parameters for maximum antimicrobial activity against

Escherichia coli and the process parameters are listed in Table 7.7 and

observed that the trend was similar to that of microencapsulated Aerva lanata

treated sample. The ANOVA tables are shown in Appendix 3.

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7.4.7 Optimized Process Parameter of Antimicrobial Activity against

Staphylococcus aureus and Escherichia coli

The numerical optimization tool of the design expert software was

used to determine the optimum values of the factors for better antimicrobial

activity against Staphylococcus aureus and Escherichia coli. The required

ranges of values for each response have been set and equal weightage was

given for all the antimicrobial activity of copper enriched herbal treated fabric

samples to get the optimum values of concentration of the medicinal herbs,

percentage of cross linking agent and curing temperature. The optimum

values of process parameters are given in Table 7.7.

Table 7.7 Optimum values of process parameters

Sl. No

Microencapsulated medicinal herbs

Concentration of extract

(in %)

Concentration of cross linking

agent (in %)

Curing temperature

(oC)

1 Aerva lanata 21 2.4 102

2 Aloe barbadensis Mill 21.19 2.44 100.03

3 Cumminum cyminum Linn 22.96 2.42 100.67

4 Tagetes erecta 21.47 2.42 100.27

5 Mentha piperita 21.28 2.36 100.4

Table 7.8 Comparison of predicted and actual antimicrobial activity with optimized process parameters

Herbs

Antimicrobial activity – zone of inhibition in mm

Aerva lanata Aloe barbadensis

Mill

Cumminum

cyminum LinnTagetes erecta Mentha piperita

S. aureus E.coli S. aureus E.coli S. aureus E.coli S. aureus E.coli S. aureus E.coli

Predicted value 30 23 30 26 30 25 29 26 30 24

Actual value 31 25 33 27 31 27 30 26 29 25

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With the optimum values of concentration of the medicinal herbs,

percentage of cross linking agent and curing temperature a trial was

performed to apply the microencapsulated copper enriched medicinal herbs.

The data given in Table 7.8 shows the comparison of predicted value obtained

from Design Expert software and actual zone of inhibition value of

antimicrobial activity with optimized parameters. In comparison with the

process parameters given in Table 7.8, it can be seen that there is a significant

improvement in the antimicrobial activity of all the microencapsulated copper

enriched medicinal herbal treated fabric sample compared to predicted values.

7.5 CONCLUSION

Methanolic extracts of copper enriched medicinal herbal plants

extracts were treated on the cotton fabric using citric acid as a cross linking

agent, at the same time direct application of extracts without crosslinking

using pad-dry-cure method and the antimicrobial activity of copper enriched

medicinal herbal treated cotton fabrics with and without crosslinking agent

and microcapsules were tested according to the standard methods for both

qualitative and quantitative analysis and the results are compared with that of

control samples. Based on the observations from this study, the crosslinking

influences by creating bonding between fibre morphology and methanolic

extracts and give a better penetration of medicinal herbs into fabric at higher

level when compared to without crosslinking medicinal herbal treated fabrics.

Microencapsulated extract of copper enriched medicinal herbs

(Aerva lanata, Aloe barbadensis Mill, Cumminum cyminum Linn, Tagetes

erecta and Mentha piperita) applied on cotton fabric were done based on Box

and Behnken experimental design by varying the process parameters like

concentration of extract, concentration of cross linking agent and curing

temperature. The microencapsulated extracts were applied by the pad-dry-

cure method and microencapsulated extracts process parameters were

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optimized using response surface methodology. The effects of these

parameters on the antimicrobial activity of treated cotton fabric material were

investigated. The zone of inhibition of treated sample increased with the

concentration of the herbal extract. The influence of curing temperature and

cross linking agent percentage on the Zone of inhibition is significant for both

the strains. The agar diffusion results show high and clear zone of inhibition

in mm against wide spectrum of human pathogenic bacterial strains of

Staphylococcus aureus and Escherichia coli.

These results were also supported by obtaining an excellent

correlation between the experimental and modeled results by formulating the

linear relationship between crosslinking agents, extract concentration and

duration of dipping time. The percentage contributions of the process

parameters including their interaction effects have also been computed to

determine their influence on the antimicrobial activity of copper enriched

medicinal herbal microencapsulated extract treated fabric.