Chapter 7 OPTIMIZATION OF EXTRACTION METHOD OF …shodhganga.inflibnet.ac.in/bitstream/10603/44915/16/16_chapter7.pdf · Microwave-assisted extraction (MAE) or simply microwave extraction

Chapter - 7

OPTIMIZATION OF EXTRACTION METHOD

OF BETULINIC ACID USING DIFFERENT

TECHNIQUES

Section TITLE Pg. No.

7.1. INSTRUMENTS AND EQUIPMENTS 113

7.2. CHEMICALS, REAGENTS AND MATERIALS 113

7.3. EXPERIMENTAL 113

7.3.1. Supercritical Fluid Extraction of betulinic acid from

Bark of D. indica

113

7.3.2. Microwave Assisted Extraction of Betulinic acid

from bark of D. indica.

115

7.3.3. Isolation and purification of BA from D. indica bark

extract obtained using SFE and MAE

117

7.3.4. Confirmation of extracted BA using FT-IR 117

7.4. RESULTS AND DISCUSSION 117-137

7.4.1. Supercritical Fluid Extraction of betulinic acid from

Bark of Dillenia indica and effect of extraction

parameters.

117

7.4.2. Microwave Assisted Extraction of betulinic acid

from bark of Dillenia indica and effect of extraction

parameters.

126

7.4.3. Comparison of SFE and MAE fractions with

standard betulinic acid

135

7.4.4. Comparison of FT-IR spectra of isolated BA and BA

procured from market.

136

7.4.5. Comparison of different extraction techniques and

yield of betulinic scid

137

7.5. CONCLUSION 138

7.6. REFERENCES 139-140

Different extraction techniques of Betulinic Acid Chapter 7

111

7. OPTIMIZATION OF EXTRACTION METHOD OF

BETULINIC ACID USING DIFFERENT TECHNIQUES

Extraction, as the term is used pharmaceutically, involves the separation of

medicinally active portions of plant or animal tissues from the inactive or inert

components by using selective solvents in standard extraction procedures. The

purposes of standardized extraction procedures for crude drugs are to attain the

therapeutically desired portion and to eliminate the inert material by treatment with a

selective solvent known as menstruum. Thus, standardization and optimization of

extraction procedures contributes significantly to the final quality of the herbal drug

[Handa SS et al., 2008]

Maceration, percolation, soxhlet, reflux and infusion are the general techniques used

for the extraction of medicinal plants and are mostly applied. Now a days, many

modern methods has been recently utilized for separation of phytoconstituents. Super

Critical Fluid extraction and Microwave Assisted extraction techniques are one of

them. In this chapter we have compared three different extraction methods for

isolation and separation of betulinic acid. Due to increasingly stringent environmental

regulations, supercritical fluid extraction (SFE) has gained wide acceptance in recent

years as an alternative to conventional solvent extraction for separation of organic

compounds in many analytical and industrial processes. In the past decade, SFE has

been applied successfully to the extraction of a variety of organic compounds from

herbs and other plants. To reduce time consuming process of traditional methods,

microwave assisted extraction gained acceptance for better isolation [Luque de Castro

and Tena, 1996].

In present chapter we have optimized two techniques supercritical fluid extraction

(SFE) as well as microwave assisted extraction for extraction of betulinic acid. The

supercritical fluid extraction (SFE) of Dillenia indica bark was carried out with pure

and modified carbon dioxide to recover the betulinic acid obtained in the collected

fractions. We investigated and analyzed a few parameters such as modifier

concentration, extraction pressure and extraction temperature. The effects of pressure

(100, 150 & 200 bar), co-solvent (methanol) content (2, 5 and 10% wt.), and


112

temperature (operation were studied in order to evaluate the applicability of SFE for

their selective and efficient production. Betulinic acid was extracted at three different

pressures keeping other two parameters constant. Same way at three different

temperature and % of modifier, betulinic acid is extracted keeping rest other

parameters constant. Results pointed out the influence of temperature, pressure and

the importance of role played by the co-solvent. At 200 bar and 50 °C, the

introduction of 10% (wt) of methanol greatly improves the yield of betulinic acid

more as compared to other conditions. % yield was calculated using developed

HPTLC method.

Microwave-assisted extraction (MAE) or simply microwave extraction is a relatively

new extraction technique that combines microwave and traditional solvent extraction.

We investigated and analyzed a few parameters such as microwave power, extraction

time and extraction temperature. Effect of three different parameters; extraction

temperature (70, 80, 90°C), microwave power (100, 150, 200 W) and extraction time

(10, 15, 20 min.) were studied. Betulinic acid was extracted at three different

temperatures keeping other two parameters constant. Same way by changing

microwave power as well as extraction time individually, betulinic acid is extracted

keeping rest other parameters constant. % extraction yield of BA calculated using

developed HPTLC method and parameters like temperature, power and extraction

time were optimized to get better extraction yield. Highest amount of betulinic acid

was obtained by extracting bark powder for 15 min at 100 W and 90°C.

Fractions obtained by SFE and MAE were collected and recrystallized to get pure

crystals of betulinic acid. Results obtained using SFE and MAE methods were

compared with those obtained by Soxhlet extraction.


113

7.1 INSTRUMENTS AND EQUIPMENTS

◘ Super critical fluid extractor with model no. JASCO 900 series, manufactured

by JASCO Inc., JAPAN with Pump (JASCO PU-980); Column Oven (JASCO-

CO-965); Back pressure regulator (JASCO-880-81); and extraction vessel.

◘ Analytical balance CITIZEN Scale CX-220, manufactured by CITIZEN Private

Ltd.; India having weighing capacity of 10mg to 220mg.

◘ Microwave extractor with model no. Synthos 3000, manufacture by Anthon

Paar, having wheaton vial (capacity 5 mL); Teflon cap and PEEK screw cap;

Rotor 64MG5 microwave synthesizer.

7.2 CHEMICALS, REAGENTS AND MATERIALS

◘ Bark powder of D. indica

◘ Standard BA, 99 % pure, Sigma-Aldrich (India).

◘ Methanol, HPLC grade and AR grade, E Merck Ltd. (India).

◘ Glacial acetic acid, AR grade, E Merck Ltd. (India).

◘ Sulphuric acid, AR grade, E Merck Ltd. (India).

◘ Anisaldehyde, AR grade, Central Drug House (P) Ltd., New Delhi (India).

◘ Chloroform, AR grade, S.D. fine Chemicals, Mumbai (India).

◘ Toluene, AR grade, S.D. fine Chemicals, Mumbai (India).

◘ Ethanol, AR grade, S.D. fine Chemicals, Mumbai (India).

◘ Pure carbon dioxide, 99.9% pure, BOC Pvt. Ltd. (India).

7.3 EXPERIMENTAL WORK

7.3.1 Supercritical Fluid Extraction of betulinic acid from Bark of D. indica

7.3.1.1 General procedure of SFE

The gaseous CO2 was cooled with cool-circulator and converted into liquid CO2

which was delivered to the extraction vessel with HPLC pump and methanol as a co-

solvent was delivered by another HPLC pump. Then, it was converted to SC-CO2

using column oven and backpressure regulator. In each 15 experimental run, 2 gm of

D. indica bark powder (prepared as per section 4.3.3.) was weighed and packed into


114

the extraction vessel with the help of glass wool. Then, SC-CO2 extraction was

performed as per the experimental parameters shown in Table 7.1. The extracts were

collected for 12 h. Fractions obtained using SFE was taken in methanol and the

collected extract volume was reduced to 10 ml with vacuum oven at 65 °C. Prepared

fractions were analyzed further using developed HPTLC method.

TABLE 7.1 Parameters for isolation of BA by SFE

Extraction Parameters Low Medium High

Co-solvent concentration (%) 2 5 10

Temperature (°C) 35 50 60

Pressure (bar) 100 150 200

7.3.1.2 Effect of Extraction Parameters in SFE

7.3.1.2.1 Effect of pressure on extraction yield

Effects of three different pressure conditions on extraction yield were investigated.

Here, the extraction pressure was varied, while modifier concentration and

temperature was kept constant. The effect of different pressure on the % yield of BA

was observed.

7.3.1.2.2 Effect of modifier on extraction yield

Three different modifier concentrations have been selected and their effects were

observed on extraction. Extraction pressure was set at 200 bar and temperature at 35

°C and modifier concentration was changed from 2-10 % v/v.

7.3.1.2.3 Effect of temperature on extraction yield

Temperature effect is too important for thermo-labile compounds. Increasing the

temperature leads to decreasing the viscosity of solvent and simultaneously increasing

the penetration power which enhance the extraction yield. Effects of three different

temperatures on extraction yield were observed. The extractions were performed by

keeping constant pressure at 200 bar and modifier concentration 10 %v/v while

changing the extraction temperature from 35-60°C.


115

7.3.1.3 Optimized conditions for isolation of BA by SFE

Different conditions applied as per section for isolation of Betulinic acid has been

optimized. Calculation of BA content in each fraction has been done as per section.

7.3.1.3.1 Reproducibility testing of SFE

Optimized conditions of supercritical fluid extractions were applied and extracts were

collected out three times. Then, the extract was analyzed with the help of developed

HPTLC method as per chapter 6. The results were compared and analyzed.

7.3.1.4 Estimation of BA in SFE fractions using developed HPTLC method

7.3.1.4.1 Calculation of % yield of BA

10 µl volume of SFE fractions and standard BA (100 µg/mL) was applied to the TLC

plate. Plates were analyzed as described in chromatographic condition. Linearity

equation of reported HPTLC method as per chapter 6 is used for quantification

purpose [Equation 7.1]. The areas of BA were kept into linearity equation and

calculate the % yield.

Linearity equation:

1.79.177459.11 EquationxY

7.3.2 Microwave Assisted Extraction of Betulinic acid from bark of Dillenia

indica.

7.3.2.1 General procedure of MAE

Sample of D. indica bark powder (5 mg) was weighed and carefully transferred into

wheaton vial each having a capacity 5 ml. For each experimental run such 20 vials

were filled with bark powder. 2 ml of methanol was added to each vial and mixed

with bark powder. Then, the vials were sealed with teflon cap and tighten by using

PEEK screw cap. Vials were placed into Rotor 64MG5 microwave synthesizer. The

extraction condition was set and the extraction process was carried out in microwave

synthesizer (Synthos 3000, Anton Paar). Effect of three different parameters;

extraction temperature, microwave power and extraction time were studied on %

extraction yield of BA as per the parameters shown in Table 7.2. The extracts from all


116

20 vials were collected and volume was reduced to 10 ml in vacuum oven at 65 °C.

Concentrated extract was analyzed and quantified with the help of reported HPTLC

method.

TABLE 7.2 Parameters for isolation of BA by MAE

Extraction Parameters Low Medium High

Extraction Temperature (°C) 70 80 90

Microwave Power (W) 100 150 200

Extraction Time (min.) 10 15 20

7.3.2.2 Effect of Extraction Parameters in MAE


Effects of three different temperature conditions on extraction yield were studied.

Here, the extraction temperature was varied, while power and time of extraction was

kept constant. The effect of different temperature on the % yield of BA was observed.

7.3.2.2.2 Effect of power on extraction yield

Three different powers have been selected and their effects were observed on

extraction. Extraction temperature was set at 90 °C, time at 15 min. and microwave

power was changed from 100-200 W.

7.3.2.2.3 Effect of time on extraction yield

Effects of three different times on extraction yield were observed. The extractions

were performed by keeping constant temperature at 90 °C and power at 100 W while

changing the extraction time from 10-20 min.

7.3.2.3 Optimized conditions for isolation of BA by MAE

Different conditions applied as per section for isolation of Betulinic acid has been

optimized. Calculation of BA content in each fraction has been done as per section.

7.3.2.3.1 Reproducibility testing of MAE

Optimized conditions of microwave assisted extractions were applied and extracts

were collected out three times. Then, the extract was analyzed with the help of


117

developed HPTLC method as per chapter 6. The results were compared and analyzed.

7.3.2.4 Estimation of BA in MAE fractions using developed HPTLC method

7.3.2.4.1 Calculation of % yield of BA

10 µl volume of MAE fractions and standard BA (100 µg/mL) was applied to the

TLC plate. Plates were analyzed as described in chromatographic condition. Linearity

equation of reported HPTLC method as per chapter 6 is used for quantification

purpose [Equation 7.1]. The areas of BA were kept into linearity equation and

calculate the % yield.

Linearity equation:

1.79.177459.11 EquationxY

7.3.3 Isolation and purification of BA from D. indica bark extract obtained using

SFE and MAE

All MAE and SFE fractions were collected using optimized condition were taken in

methanol. All the fractions were combined into beaker. Extract were concentrated on

boiling water bath until semisolid mass is obtained. The semisolid yellowish mass

was washed with methanol three times. After washing, the residue was kept in

refrigerator for 2 days. At last, the white crystalline compound was obtained. Collect

the crystalline compound and stored into refrigerator.

7.3.4 Confirmation of extracted BA using FT-IR

Obtained crystals obtained using SFE, MAE were checked and confirmed using FT-

IR method. Dried spectroscopic grade KBr powder was taken and triturates it. A KBr

spectrum as a blank was taken. Then, mix the extracted BA crystalline powder with

standard spectroscopic grade KBr powder in 1:100 ratio and take their spectra.

7.4 RESULTS AND DISCUSSION

7.4.1 Supercritical Fluid Extraction of betulinic acid from Bark of D. indica

Total 12 fractions were collected at each hour fraction obtained was collected in

methanol and prepared as per section 7.3.1.1. Each fraction was analyzed using

developed HPTLC method mentioned in chapter 6. Chromatogram obtained is


118

mentioned in Figure 7.1 which is compared with standard betulinic acid

chromatogram (Figure 7.2).

FIGURE 7.1 HPTLC chromatogram of Supercritical fluid extract of BA at optimized

condition (Rf 0.70)

FIGURE 7.2 HPTLC chromatogram of standard BA (Rf 0.70)

7.4.1.1 Effect of Extraction Parameters in SFE

The main aspects are taken into consideration for SFE method development is

extraction optimization. The optimum values for the different variables influencing

the SFE extractions significantly enhance the recovery or extraction yield of betulinic

acid. The effect of parameters and yield obtained has been reported here.


119

7.4.1.1.1 Effect of pressure on extraction yield

If the extraction temperature is increased at a constant pressure, the density of

supercritical CO2 will decrease. The saturation pressure of solute in SCF increases

with the increase of temperature, which improves the solubility. It is well known that

with the increase of pressure, the density of SC-CO2 increases, and the solubility of

solute increases which enhance the extraction efficiency. Results indicates that BA

extraction yield were increased with increasing pressures. Maximum yield of BA was

obtained at 200 bar (Figure 7.3, 7.4).

FIGURE 7.3 HPTLC Densitometric chromatogram of BA at different pressure in

SFE with 5% MeOH and 35 °C temperature

FIGURE 7.4 Supercritical fluid extract containing BA at 200 bar/35 °C/5%

200 bar


120

7.4.1.1.2 Effect of modifier on extraction yield

The good extraction yield was obtained with greater modifier concentration. Methanol

added to the supercritical CO2 increases the polarity of the fluid. The modifier exerts

its effect mainly in two basic ways: by interacting with the analyte complex to

promote rapid desorption into the supercritical fluid, and by enhancing the solubility

properties of supercritical CO2 [Luque de Castro and Tena, 1996]. Various

concentration of methanol used exhibited different effect in changing the polarity and

thus had diverse effect on the solubility enhancement of the betulinic acid. In this

study, the results indicated that the optimal methanol concentration for extraction 10%

[Ghasemi E et al., 2007]. Figure 7.5 reveals that the usages of 2 and 5 %v/v modifier

concentration give little effect on extraction yield while, 10 %v/v modifier

concentration gives significant increase in % yield of BA. Further increasing the

modifier concentration above 10 %v/v increases extraction time for complete

extraction of BA. It also requires more consumption of power and solvent and there is

also miscibility problem with super critical carbon dioxide. Hence, considering all the

aspects the 10%v/v modifier concentration was found optimal for getting maximum

extraction yield.

FIGURE 7.5 HPTLC Densitometric chromatogram of BA at different concentration

of modifier in SFE with 200 bar pressure and 35 °C temperature


121

FIGURE 7.6 Supercritical fluid extract containing BA at 10%/200bar/35 °C


The influence of temperature on extraction is more difficult to predict than that of

pressure, because of its two counter effects on the yield. First, the temperature

elevation decreases the density of CO2, leading to a reduction in the solvent power to

dissolve the solute. Second, the temperature rise increases the vapor pressure of the

solutes, bringing about the elevation in the solubility of chemical constituents in SF-

CO2. In general, solvent strength and diffusivity can both be increased by raising the

extraction temperature. The overall extraction effect of supercritical fluids usually

follows the competition between the increasing in solute of compounds and the

reduction in SC-CO2 density due to the rise in temperature. Figure 7.7 shows the

effect of temperature on extraction yield. On the basis of % yield found in all

conditions the optimal condition of temperature was found to be 50°C for maximum

extraction yield. Temperature more than 50°C shows detrimental effect on extraction

yield [Yin JZ et al., 2005; Salgin. U et al., 2006; Ozkal SG, 2005; Louli V et al.,

2004]

10%


122

FIGURE 7.7 HPTLC Densitometric chromatogram of BA at different temperature in

SFE with 200bar pressure and 10% MeOH

7.4.1.2 Optimized conditions for isolation of BA by SFE

Amount of betulinic acid calculated. It was observed from the reported data in section

7.4.1.1 that at 50 °C keeping 200 bar pressure and 10 % concentration of modifier

highest yield is obtained. SFE method was applied at optimized parameters and

further used for extraction of betulinic acid. Results obtained as per optimized

parameters of supercritical fluid extraction has been mentioned in Table 7.3.

FIGURE 7.8 Supercritical fluid extract containing BA at 50 °C/200bar/10%

50 °C


123

TABLE 7.3 SFE results of BA by HPTLC

SR. NO. BETULINIC ACID Rf AREA

1 Supercritical fluid extract

containing BA

0.70 15,610

2 Standard BA (1000 ng/spot) 0.70 12,500

7.4.1.3 Reproducibility testing of SFE

SFE fractions were checked for reproducibility and reproducible results were obtained

which is shown in Figure 7.9 (Table 7.4). From the reported data it has been observed

that the developed supercritical fluid extraction method is found to be reproducible.

FIGURE 7.9 HPTLC Densitometric chromatogram of BA reproducible result in SFE

TABLE 7.4 Reproducible results of BA extract obtained by SFE

Run Co-solvent

concentration

(%)

Extraction

temperature

(°C)

Extraction

pressure

(bar)

%w/w % RSD

1 10 50 200 0.71 1.75

2 10 50 200 0.69

3 10 50 200 0.69


124

7.4.1.4 Amount of BA in SFE fraction using HPTLC method

SFE extract containing BA and standard BA (100 µg/mL) was applied on the TLC

plate and the % yield obtained by quantifying amount of betulinic acid in SFE

fraction at different concentration, temperature and pressure using HPTLC method is

mentioned here. It has been observed from obtained results that at 2 % concentration

50 °C temp and 150 bar pressure yield obtained was 0.339 %w/w (Table 7.5). By

keeping 5% concentration, 60 temperature and 200 bar pressure yield obtained was

0.438 %w/w which has been mentioned in Table 7.6. At 10% concentration, 50 °C

temp and 200 bar pressure showed highest yield 0.690 %w/w (Table 7.7).

TABLE 7.5 SFE results of BA using 2% concentration of solvent

Batch

No.

Co-solvent

concentration

(%)

Extraction

temp (℃)

Extraction

pressure

(bar)

% w/w

in bark

1 2 35 200 0.313

2 2 35 150 0.182

3 2 35 100 0.216

4 2 50 100 0.181

5 2 50 150 0.339

6 2 50 200 0.189

7 2 60 100 0.171

8 2 60 150 0.187

9 2 60 200 0.161

TABLE 7.6 SFE results of BA using 5% concentration of solvent

Batch

No.

Co-solvent

concentration

(%)

Extraction

temp (℃)

Extraction

pressure

(bar)

% w/w

in bark

1 5 35 100 0.287

2 5 35 150 0.198

3 5 35 200 0.107

4 5 50 100 0.256

5 5 50 150 0.339

6 5 50 200 0.119

7 5 60 100 0.412

8 5 60 150 0.231

9 5 60 200 0.438


125

TABLE 7.7 SFE results of BA using 10% concentration of co-solvent

Batch

No.

Co-solvent

concentration

(%)

Extraction

temp (℃)

Extraction

pressure

(bar)

% w/w

in bark

1 10 35 100 0.310

2 10 35 150 0.433

3 10 35 200 0.261

4 10 50 100 0.524

5 10 50 150 0.139

6 10 50 200 0.690

7 10 60 100 0.218

8 10 60 150 0.475

9 10 60 200 0.324

7.4.1.5 HPTLC Plate showing fractions collected using SFE

Derivatized HPTLC plate of collected fractions using supercritical fluid extraction

from bark of D. indica at different optimized conditions is shown in Figure 7.10.

FIGURE 7.10 Derivatized HPTLC plate showing BA by SFE at different time

interval at 10% MeOH, 50 °C temperature and 200 bar pressure.

[1,2: 1 hr extract; 3,4: 2 hr extract; 5,6: 3 hr extract; 7,8: 4 hr extract; 9,10: 5 hr extract; 11: Standard

BA; 12,13:6 hr extract; 14,15: 7 hr extract; 16,17: 8 hr extract; 18,19: 9 hr extract; 20,21: 10 hr extract;

22,23:11 hr extract; 24,25: 12 hr extract]


126

7.4.2 Microwave Assisted Extraction of betulinic acid from bark of Dillenia

indica

Collected fractions from 20 vials were combined and prepared as per section 7.3.2.1.

and have been analyzed using developed HPTLC method. Each fraction of MAE was

analyzed using developed HPTLC method mentioned in chapter 6. Chromatogram of

fractions collected by MAE obtained is mentioned in Figure 7.11 which is compared

with standard betulinic acid chromatogram (Figure. 7.12).

FIGURE 7.11 HPTLC chromatogram of Microwave assisted extract of BA at

optimized condition (Rf 0.70)

FIGURE 7.12 HPTLC chromatogram of standard BA (Rf 0.70)


127

7.4.2.1 Effect of Extraction Parameters in MAE

Three different factors were analyzed to get good amount of betulinic acid from bark

powder. Microwave power and temperature are interrelated because high microwave

power can bring up the temperature of the system and result in the increase of the

extraction yield until it becomes insignificant or declines [Hu Z et al., 2008; Xio W et

al., 2008; Chemat S et al., 2005]. It is known that the temperature is controlled by

incident microwave power that controls the amount of energy provided to the matrix,

which is converted to heat energy in the dielectric material. At high temperatures the

solvent power increases because of a drop in viscosity and surface tension, facilitating

the solvent to solubilize solutes, and improving matrix wetting and penetration

[Mandal V et al., 2007; Li J et al, 2010; Khajeh M et al., 2009]. In addition, when

MAE is performed in closed vessels, the temperature may reach far above the boiling

point of the solvent, leading to better extraction efficiency by the desorption of solutes

from actives sites in the matrix [Eskilsson CS & Bjorklund E, 2000]. Microwave

power is directly related to the quantity of sample and the extraction time required.

However, the power provides localized heating in the sample, which acts as a driving

force for MAE to destroy the plant matrix so that the solute can diffuse out and

dissolve in the solvent. Therefore, increasing the power will generally improve the

extraction yield and result in shorter extraction time [Hu Z, 2008, Xiao W, 2008].

In MAE the period of heating is also another important factor to be

considered. Extraction times in MAE are very short compared to conventional

techniques and usually vary from a few minutes to a half-hour, avoiding possible

thermal degradation and oxidation, which is especially important for target

compounds sensitive to overheating of the solute–solvent system [Chan CH et al.,

2011, Al-Harahshed M et al., 2004]. Overheating occurs because of the high dielectric

properties of the solvent, especially ethanol and methanol, and further dilution with

water that increases the heat capacity of the solvent combination [Routray W & Orsat

V, 2011]. Higher extraction time usually tends to increase the extraction yield.

However, this increase was found to be very small with longer time [Wang Y et al.,

2008].


Increasing the temperature leads to increasing the vapour pressure in closed system

which improved extraction efficiency since desorption of chemical constituent from


128

sample matrix will be facilitated. Increasing the temperature also shows the

decreasing surface tension and viscosity of solvent which will improve sample

wetting and matrix penetration respectively. Therefore, BA extraction efficiency was

increase with increasing temperature which is depict in figure 26. Maximum yield of

betulinic acid was obtained at 90 °C. (Figure 7.13, 7.14)

FIGURE 7.13 HPTLC Densitometric chromatogram of BA at different temperature

in MAE with 200 W and 15 min.

FIGURE 7.14 Microwave assisted extract containing BA at 90 °C/200W/15min.

90 °C


129

7.4.2.1.2 Effect of power on extraction yield

The good extraction yield was obtained at 100 W. Figure 28 indicate that the increase

in power greater than 100 leads to detrimental effect on % yield but it does not shows

linear effect. Hence, considering all the aspects 100 W power was found optimal for

getting maximum extraction yield. (Figure 7.15, 7.16)

FIGURE 7.15 HPTLC Densitometric chromatogram of BA at different power in

MAE with 90 °C and 15 min.

FIGURE 7.16 Microwave assisted extract containing BA at 100W/90°C/15min.

100 W


130

7.4.2.1.3 Effect of time on extraction yield

Figure 30 reveals the effect of time on extraction yield. On the basis of % yield found

in all conditions the optimal condition of time was found to be 15 min. for maximum

extraction yield. Time more than 15 min. showed detrimental effect on extraction

yield since the methanol heat up tremendously on longer exposure. (Figure 7.17, 7.18)

FIGURE 7.17 HPTLC Densitometric chromatogram of BA at different time in MAE

with 90 °C and 100 W.

FIGURE 7.18 Microwave assisted extract containing BA at 15 min./100W/90°C

15 min.


131

7.4.2.2 Optimized conditions for isolation of BA by MAE

Amount of betulinic acid calculated. It was observed from the reported data in section

7.4.2.1. that highest yield was obtained by extracting bark powder for 15 min at 90 °C

keeping 100 W microwave power. MAE method was applied out at optimized

parameters and further used for extraction of betulinic acid. Results obtained as per

optimized parameters of microwave assisted extraction have been mentioned in Table

7.8.

TABLE 7.8 MAE Results of BA by HPTLC

SR. NO. BETULINIC ACID Rf AREA

1 Microwave assisted extract

containing BA

0.71 18,800

2 Standard BA (1000 ng/spot) 0.70 12,500

7.4.2.3 Reproducibility Results of MAE fractions

MAE fractions were checked for reproducibility and reproducible results were

obtained which is shown in Figure 7.19 (Table 7.9). From the reported data it has

been observed that the developed microwave assisted extraction method is found to be

reproducible.

FIGURE 7.19 HPTLC Densitometric chromatogram of BA reproducible result in

MAE


132

TABLE 7.9 Reproducible results of BA extract obtained by MAE

Run Extraction

Temperature

(°C)

Microwave

Power

(W)

Extraction

Time (min.) %w/w % RSD

1 90 100 15 0.88

2.35 2 90 100 15 0.91

3 90 100 15 0.92

7.4.2.4 Amount of BA in MAE fractions using HPTLC method

MAE extract containing BA and standard BA (100 µg/mL) was applied to the TLC

plate and the % yield obtained by quantifying amount of betulinic acid in MAE

fraction at different parameters used such as temperature, microwave power and

pressure using HPTLC method is mentioned here. It has been observed from obtained

results that at 70 °C temp keeping microwave power 100 W for 15 min yield obtained

was 0.57 %w/w (Table 7.10). By keeping 60°C temperature, 150 W and 15 min

extraction time yield obtained was 0.66% which has been mentioned in Table 7.11. At

90 °C temp., 100 W for 15 min. highest yield 0.91% w/w (Table 7.12).

TABLE 7.10 MAE results of BA at Extraction temperature (70°C)

Batch

No.

Extraction

Temperature

(°C)

Microwave

Power

(W)

Extraction

Time

(min.)

% w/w in

bark

1 70 100 10 0.21

2 70 100 15 0.57

3 70 100 20 0.35

4 70 150 10 0.55

5 70 150 15 0.29

6 70 150 20 0.53

7 70 200 10 0.24

8 70 200 15 0.52

9 70 200 20 0.31


133


Batch

No.

Extraction

Temperature

(°C)

Microwave

Power

(W)

Extraction

Time

(min.)

% w/w in

bark

1 80 100 10 0.63

2 80 100 15 0.51

3 80 100 20 0.61

4 80 150 10 0.43

5 80 150 15 0.66

6 80 150 20 0.49

7 80 200 10 0.57

8 80 200 15 0.37

9 80 200 20 0.56


Batch

No.

Extraction

Temperature

(°C)

Microwave

Power

(W)

Extraction

Time

(min.)

% w/w in

bark

1 90 100 10 0.53

2 90 100 15 0.91

3 90 100 20 0.79

4 90 150 10 0.83

5 90 150 15 0.67

6 90 150 20 0.80

7 90 200 10 0.48

8 90 200 15 0.71

9 90 200 20 0.69


134

7.4.3.4.1 HPTLC Plate showing fractions collected using MAE

Derivatized HPTLC plate of collected fractions using microwave assisted extraction

of betulinic acid from bark of D. indica at different optimized conditions is shown in

Figure 7.20.

FIGURE 7.20 Derivatized HPTLC plate showing BA by MAE

at different time interval.

[1,7,15 : Standard BA; 2,3: 5 min. extract; 4,5: 10 min. extract; 6,8 : 15 min. extract;

9,10 : 20 min. extract; 11,12: 25 min. extract; 13,14: 30 min. extract]


135

7.4.3 Comparison of SFE and MAE fractions with standard betulinic acid

Figure 7.21 shows HPTLC Densitometric chromatogram (3D) of BA at optimized

SFE and MAE conditions which is matching with chromatogram of standard betulinic

acid.

FIGURE 7.21 HPTLC Densitometric chromatogram (3D) of BA

at optimized SFE and MAE conditions

[1,2 : Supercritical fluid extract of BA at optimized condition; 3,4 : Microwave

assisted extract of BA at optimized condition; 5: Standard BA]


136

7.4.4 Comparison of FT-IR spectra of isolated BA and BA procured from

market

Obtained crystals of BA as per section 7.4.4. has been taken for reconfirmation using

FT-IR spectroscopy. FT-IR spectra of isolated BA by different extraction methods as

well as BA procured from market is shown in Figure 7.22 & 7.23 respectively.

FIGURE 7.22 Recorded FT-IR spectra of extracted BA from D. indica bark.

FIGURE 7.23 FT-IR spectra of BA standard


137

7.4.5 Comparison of different extraction techniques and yield of betulinic acid

Table 7.13 shows comparison of developed super critical fluid extraction and

microwave assisted extraction with conventional soxhlet extraction method mentioned

in chapter 5 (Section 5.3.1). Extracts obtained using each method yielded betulinic

acid in form of crude crystals. Purity of betulinic acid obtained using soxhlet

extraction was found to be average which can overcome by SFE and MAE methods.

After soxhlet extraction, extract needs further separation of betulinic acid which can

be done by column chromatography. While in case of SFE and MAE, pure crystals

can be recrystallized simply using methanol.

TABLE 7.13 Comparison of Extraction techniques

Soxhlet

extraction SFE MAE

Presence of BA

in obtained

extract

Average

(needs further

separation)

Good Quantity

(further

recrystallized)

Good quantity

(Further

recrystallized)

Time

For Separation

Very high

(In Days) 12 h or more 30 min Max.

Organic

solvent

consumption

High Less Average

Eco-friendly No Yes No

Yield of BA

(% w/w) 0.28- 0.36 0.69 0.91


138

7.5 CONCLUSION

In the present study, two advanced techniques i.e., SFE and MAE were designed and

optimized for the extraction of BA from D. indica bark which has been compared

with traditional conventional method i.e., soxhlet extraction. The results presented in

this work indicated that developed SFE and MAE was feasible for extraction of

betulinic acid from bark of Dillenia indica. Simple and reproducible method for

isolation of betulinic acid in pure form has been described here.

HPTLC analysis results of SFE and MAE fractions proved the method is significant

and useful for better extraction of betulinic acid. The percentage yield of BA obtained

by soxhlet extraction, SFE, MAE were 0.28-36 % w/w, 0.69 %w/w and 0.91 %w/w

respectively.

All three methods are having their own significance and their own advantages and

disadvantages. In SFE, CO2 is the solvent of choice and consumption of toxic organic

solvent (modifiers) is also less (0.34 mL/min.) which creates the extraction process

more eco-friendly. MAE showed highest yield of BA as compared to other methods

but in MAE besides the BA, interference of other chemical constituent is higher that

was also obtained in SFE but less interference was observed while in case of simple

precipitation directly pure crystals were obtained which can be used directly for

further evaluation. The SFE extract shows slightly higher recovery of BA since it is

totally free of solvents and very pure.

Soxhlet extraction technique is advantageous than other two because SFE and MAE

require specialized equipments but it requires further separation in order to separate

BA in pure form. This can easily be achieved in other two methods. One more

disadvantage of conventional method required more amount of chemicals and

reagents as compared to other two methods. This disadvantage is overcome by other

two methods.

Compared with MAE, SFE have two major advantages:

i. The supercritical fluids dissolving properties depends on its density, which is

adjustable by changing the pressure and temperature;


139

ii. The mass transfer is higher in case of SFE since supercritical fluid has a higher

diffusion coefficient, lower viscosity and surface tension than a liquid solvent.

Hence, the SFE technique is superior to MAE because;

i. The SFE technique gives extract with minimum interference of other component

ii. Easily transferred from lab-scale to industry

iii. Sustainable

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