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2 Isolation and Purification of 3,5-Diprenyl-4-Hydroxycinnamic Acid (DHCA) in Brazilian Propolis

2 Isolation and Purification of 3,5-Diprenyl-4-Hydroxycinnamic Acid (DHCA) in Brazilian Propolis

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2



Green Fluids Extraction and Purification of Bioactive Compounds…



71



DHCA Extracted (mg/g of solid)



100



80



60



40



EA 76oC

N-hexane 69oC

EtOH 78oC

H2O 100oC



20



0



0



5



10



15



20



25



Extraction Time (hr)

Fig. 2.1 Four Soxhlet solvent extractions of the DHCA from Brazilian propolis lump (Reprinted

from Ref. [18]. With kind permission of © Elsevier)



ultrasonicated in the 1,200 g ethyl acetate for a period of 1.5, 2.5, and 3.5 h, operated

at 308, 328, and 348 K, respectively. For the hot-pressurized extractions, 10 g of propolis powder dissolved in 1,200 g of ethyl acetate was loaded in an autoclave extractor,

and the solution was purged by 4.5-bar nitrogen for 10 min to strip off the oxygen

entrapped in the solution. The experiments were carried out and controlled at 4.5-bar

and 300-rpm agitation speed with temperature ranging from 348 to 388 K. All the

extracts were filtered through the Whatman no.1 filter paper and weighted to calculate

the total yield as well as the recovery and the purity of DHCA in the extracts.

Figure 2.1 indicates the effect of extraction time on the amount of DHCA

extracted by four Soxhlet extractions. After 16 h of extraction, the maximal DHCA

in the propolis extract was 91.9 mg/g, obtained with 120 mL of ethyl acetate Soxhlet

extraction of 1-g propolis lump. This was recognized as a 100% recovery of DHCA

from this Brazilian propolis lump. For the hot-pressurized extraction, the recovery

of DHCA attained 88.5%, but the extraction time was reduced from 16 down to

2.5 h. In addition, reduced standardized concentration factors (i.e., a = b/bSoxhlet < 1.0)

revealed that the DHCA purity in propolis extracts was less than that of the Soxhlet

ethyl acetate extraction (16.9 wt%). For the ultrasonic extraction, the recovery of

DHCA was only 75%, and the DHCA purity was also less than that of the Soxhlet

ethyl acetate extraction.



2.2.2



Purification and Identification of 3,5-Diprenyl-4Hydroxycinnamic Acid (DHCA)



Two methods, namely, liquid-liquid solvent partition and normal-phase column

chromatography were employed to purify DHCA from the propolis extracts. In the



72



C.-R. Chen et al.



first method, the waxes were removed by n-hexane washing firstly and the raffinate

was then dried to redissolve in methanol. Contrarily, the second method was adopted

to avoid n-hexane and methanol partitions. The final residue was then collected by

removing the supernatant using a centrifuge at 8,000 rpm for 10 min. The residue

was dissolved in a mixture of 7:3 (v/v) n-hexane and ethyl acetate (EA) to form a

crude solution. The crude solution was manually loaded and fractionated on a silica

gel 60 column (2.5-cm I.D.), eluted by a mixed mobile phase consisting of n-hexane

and EA in different volume ratios. Every 10-mL eluent was collected at the flow rate

of 2.5 mL/min. The sample containing 95-wt% DHCA was dissolved in CDCl3 and

analyzed by a 400-Hz 1H-NMR spectrophotometer (Varian, Mercury, USA).

Experimental data (Table 2.1) resulted from these column purifications of the

propolis extracts with solvent pretreatment (i.e., first method) indicated DHCA

purities of the purified products as 95 wt%, except for the Soxhlet extract (91 wt%).

The highest total recovery by this procedure was only 4.2% for the purified product.

Major losses of DHCA were found in the n-hexane layer (20%) and methanol layer

(60%). Table 2.2 lists experimental results of purified DHCA without solvent pretreatment (i.e., second method). Solely the purified products of those SC-CO2

extracts contained 95 wt% DHCA, and the largest total recovery could reach 9.4%,

which is 2.3 times to that of the hot-pressurized extract in the first method. These

SC-CO2 extracts containing 40 wt% DHCA are easy to be purified by column

purification alone.



2.2.3



Quantification of 3,5-Diprenyl-4-Hydroxycinnamic

Acid (DHCA)



A Waters HPLC system, which comprised a 600E multisolvent delivery pump, a

717 plus autosampler, a 486 UV/Vis detector, and Millennium 2010 system manager software, was employed to analyze the extracts. (10 mL) Samples were filtered

through a 0.45-mm polyvinylidene fluoride (PVDF) membrane (Millipore, USA)

and were injected into a C18 reversed-phase column (4.6 × 250 mm, 5U, Hichrom,

UK) to quantify the DHCA of the extracts. The column temperature was controlled

at 308 K, and the UV absorption was detected at a wavelength of 280 nm. A twosolvent gradient mobile phase comprising 55% methanol (A) and 80% methanol

(B) in 0.5% acetic acid aqueous solution was used for this analysis. A calibration

curve was established by linear regression between the area under the UV absorption curve and the concentration of the samples. A protocol sample containing

95 wt% DHCA was isolated from the 61 to 75 fractions collected and was used as

the standard for this study. The purity of this standard was an averaged value verified by the HPLC method, and the concentration ranged from 20 to 620 mg/mL. The

R2 coefficients exceeded 0.99, and the limits of detection in these analyses were

3,220 ng/mL. Figure 2.2 represents HPLC spectra of samples including the 95 wt%

DHCA spectrum and its chemical shift pattern of 1H-NMR, which is exactly corresponding to that reported by Bohlmann et al. [21].



Soxhlet: 16-h EA; HPE: 2.5-h, 368-K hot-pressurized EA; Sonic: 2.5-h, 348-K ultrasonic-EA; SCF1: 323-K, 207-bar, 6-wt% EA + 475 L CO2; SCF2: 333-K, 207bar, 6-wt% EA + 475-L CO2; SCF: 323-K, 207-bar, 475-L CO2

WDHCA the DHCA weight in the extracted propolis, Wpropolis the extracted weight of propolis, PDHCA the DHCA purity of the extracted propolis = 100% × (WDHCA/

Wpropolis), WPD the purified weight of DHCA, WPT total weight of the purified product, PPD the DHCA purity of the purified product = 100% × (WPD/WPT), RPD recovery of 95 wt% DHCA for the first purification process = 100% × (WPD/WDHCA), RT total recovery of 95 wt% DHCA = 100% × (WPD/WDHCA,Soxhlet), Lhexane the weight

loss of DHCA in n-hexane = (WDHCA-WPD-LMeOH-Lothers), LMeOH the weight loss of DHCA in MeOH = (WDHCA-WPD-Lhexane-Lothers), Lothers the weight loss of DHCA in

others = (WDHCA-WPD-Lhexane-LMeOH), LP the percentage of DHCA lost in each step = 100% × (L/WDHCA)



Soxhlet 91.9±0.90 543.4±3.76 16.9±0.20 3.82±0.12 4.20±0.14 91±0.3 4.16±0.09 4.16±0.09 18.75±2.03

56.79±1.24

12.54±2.49

[20.40±2.21]

[61.79±1.35]

[13.67±2.84]

HPE

83.2±0.35 506.9±5.00 16.4±0.23 3.72±0.11 3.92±0.13 95±0.4 4.47±0.11 4.05±0.12 16.26±1.22

50.76±1.08

12.41±2.06

[19.53±1.47]

[61.01±1.30]

[14.94±2.54]

Sonic

69.0±0.35 432.4±0.55 16.0±0.06 3.14±0.12 3.31±0.15 95±0.4 4.56±0.20 3.42±0.13 13.42±0.88

41.61±1.23

10.83±2.58

[19.44±1.28]

[60.31±1.78]

[15.69±3.66]

SCF1

12.6±0.12 30.7±0.34 41.2±0.04 0.72±0.05 0.76±0.07 95±0.5 5.71±0.34 0.78±0.05 2.86±0.42

7.12±0.81

1.90±0.74

[22.70±3.33]

[56.51±6.43]

[15.08±5.87]

SCF2

12.7±0.04 31.1±0.16 40.8±0.01 0.68±0.06 0.72±0.05 95±0.2 5.34±0.45 0.74±0.07 2.66±0.56

7.98±0.92

1.39±0.27

[20.94±4.41]

[62.83±7.24]

[10.94±2.13]

SCF

3.7±0.10

8.17±0.85 45.3±0.10 0.21±0.02 0.22±0.02 95±0.3 5.67±0.39 0.23±0.02 0.72±0.13

2.41±0.27

0.36±0.09

[19.46±3.51]

[65.14±7.29]

[9.73±2.43]



Table 2.1 Experimental data purifying DHCA from propolis extracts by the first method with solvent pretreatments (Reprinted from Ref. [18]. With kind permission

of © Elsevier)

Extracts

Purified Products

DHCA Loss

Recovery

(%)

LMeOH (mg/g)

Lothers (mg/g)

Wpropolis

PDHCA

WPD

WPT

PPD

Lhexane (mg/g)

WDHCA

RT

(mg/g)

(wt%)

(mg/g)

(mg/g)

(wt%) RPD

[LPhexane (%)]

[LPMeOH (%)]

[LPothers (%)]

Method (mg/g)



2

Green Fluids Extraction and Purification of Bioactive Compounds…

73



91.9 ± 0.90



83.2 ± 0.35



69.0 ± 0.35



12.6 ± 0.12



12.7 ± 0.04



3.7 ± 0.10



Soxhlet



HPE



Sonic



SCF1



SCF2



SCF



8.17 ± 0.85



31.1 ± 0.16



30.7 ± 0.34



432.4 ± 0.55



506.9 ± 5.00



543.4 ± 3.76



Wpropolis (mg/g)



45.3 ± 0.10



40.8 ± 0.01



41.2 ± 0.04



16.0 ± 0.06



16.4 ± 0.23



16.9 ± 0.20



PDHCA (wt%)



2.56 ± 0.21



8.65 ± 0.43



8.64 ± 0.32



32.14 ± 1.70



43.14 ± 2.13



42.74 ± 2.10



WPD (mg/g)



2.69 ± 0.22



9.11 ± 0.41



9.09 ± 0.31



41.21 ± 1.71



56.03 ± 2.15



60.20 ± 2.09



WPT (mg/g)



95 ± 0.6



95 ± 0.4



95 ± 0.5



78 ± 0.5



77 ± 0.6



71 ± 1.2



PPD (wt%)



69.09 ± 3.81



67.99 ± 3.17



68.55 ± 1.89



46.63 ± 2.70



51.87 ± 2.34



46.49 ± 1.83



RPD (%)



2.79 ± 0.23



9.41 ± 0.47



9.40 ± 0.35



34.97 ± 1.85



46.94 ± 2.32



46.49 ± 1.83



RT (%)



49.16 ± 0.90

[53.49 ± 0.48]

40.06 ± 0.35

[48.14 ± 0.23]

36.83 ± 0.35

[53.38 ± 0.25]

3.96 ± 0.12

[31.43 ± 0.68]

4.05 ± 0.04

[31.99 ± 0.22]

1.14 ± 0.10

[30.81 ± 2.02]



[LPDHCA (%)]



Soxhlet: 16-h EA; HPE: 2.5-h, 368-K hot-pressurized EA; Sonic: 2.5-h, 348-K ultrasonic EA; SCF1: 323-K, 207-bar, 6-wt% EA + 475-L CO2; SCF2: 333-K,

207-bar, 6-wt% EA + 475-L CO2; SCF: 323-K, 207-bar, 475-L CO2

WDHCA the DHCA weight in the extracted propolis, Wpropolis the extracted weight of propolis, PDHCA the DHCA purity of the extracted propolis = 100% × (WDHCA/

Wpropolis), WPD the purified weight of DHCA, WPT total weight of the purified product, PPD the DHCA purity of the purified product = 100% × (WPD/WPT), RPD recovery of 95 wt% DHCA for the first purification process = 100% × (WPD/WDHCA), RT total recovery of 95 wt% DHCA = 100% × (WPD/WDHCA,Soxhlet), LDHCA the weight

loss of DHCA in experiments = (WDHCA-WPD), LPDHCA the percentage of DHCA lost in experiments = 100% × (WDHCA-WPD)/WDHCA



WDHCA (mg/g)



Method



Table 2.2 Experimental data purifying DHCA from propolis extracts by the second method without solvent pretreatments (Reprinted from Ref. [18]. With kind

permission of © Elsevier)

Extracts

Purified products

DHCA recovery

DHCA loss

LDHCA (mg/g)



74

C.-R. Chen et al.



2



Green Fluids Extraction and Purification of Bioactive Compounds…



a



75



0.80

0.70

0.60



AU (V)



0.50



DHCA



0.40

0.30

0.20



a

b

c

d

e

f



0.10

0.00



0.071

0.000



1.284

1.251



100.0



1.795

1.781



80.0



3.362

3.343



AC

Pulse Sequence: s2pul



60.0

Minutes



5.313

5.309

5.306



40.0



6.309

6.269



20.0



7.264

7.203



b



7.713

7.674



0.0



O

OH



HO



8



7

0.99



1.85



6

1.00



5

0.71



2.04



4



3

3.82



2



1



0



ppm



12.58



Fig. 2.2 (a) HPLC chromatograms of the extracts detected at 280 nm [a: Soxhlet-EA; b:

SC-CO2 + 2-wt% n-hexane; c: SC-CO2 + 6-wt% EA; d: SC-CO2; e: 80-wt% DHCA; f: purest

95-wt% DHCA]. (b) A 400-Hz 1H NMR spectrum of the 95-wt% DHCA [d : 7.69 (1H, d,

J = 15.9 Hz), 7.20 (2H, s), 6.29 (1H, d, J = 15.9 Hz), 5.31 (2H, br, t), 3.35 (2H, d, J = 7.2 Hz), 1.79

(6H, s), and 1.78 (6H, s)] (Reprinted from Ref. [18]. With kind permission of © Elsevier)



C.-R. Chen et al.



76



2.3



2.3.1



Green Fluid Extraction of 3,5-Diprenyl-4Hydroxycinnamic Acid (DHCA) from Brazilian Propolis

Sensitivity Test of Supercritical Carbon Dioxide

(SC-CO2) Extractions



Propolis lumps were ground into 2-mm particles using a blade-type grinder and

then collected by sieving through a 10-mesh international-type stainless steel screen

before use. The maximum recovery of DHCA in Brazilian propolis was obtained by

Soxhlet ethyl acetate extraction during a period of 16 h. Figure 2.3 presents the

SC-CO2 extraction that was used by You et al. [23]. Before the extraction, 10 g of

ground propolis powder and 30 g of steel beads were uniformly loaded into an

extractor (75 mL, L/D = 30) (8); a 5-cm thickness of glass wool was placed on both

the top and the bottom of the extractor to prevent the entrainment of propolis particles, and 200 mL of 95% ethanol was loaded into the absorber (750 mL, L/D = 10)

(11), which acted as a absorbent. Then, liquid CO2 was pumped using a highpressure pump (CM-3200, Thermo Separation Products, USA) and flowed into the

extractor at a constant flow rate of 10 mL/min after preheating. The extraction pressure varied from 138 to 276 bar and was regulated using a back-pressure regulator

(26–1721, Tescom, USA) (9–1), and the extraction temperature ranged from 308 to

333 K, controlled by a PI-type controller. The pressure of the absorber was set to

50 bar, and another back-pressure regulator (9–2) was used to separate CO2 from the

extracts that were collected at ambient temperature. The consumed CO2 volume was

measured using a wet gas meter (TG3, Ritter, Germany) (13). Furthermore, the

amount of ethyl acetate, from 0 to 6 wt%, was weighed and preloaded in the extractor as a cosolvent.

The 91.9 mg/gpropolis of DHCA was yielded via Soxhlet ethyl acetate extraction

from Brazilian propolis lumps. Table 2.3 presents the results of Soxhlet and

SC-CO2 extractions of DHCA. It states that only few DHCA could be extracted by

adding n-hexane as cosolvent and both recovery and purity of DHCA were lower

than that by adding ethyl acetate. Furthermore, the standardized concentration factors of DHCA of modified SC-CO2 extracts obtained by addition of ethyl acetate

and n-hexane were 2.59 and 2.14, respectively. Figure 2.4 shows the addition of

EA on the recovery and purity of DHCA obtained from SC-CO2 extractions. The

recovery of DHCA increased with the consumption of CO2 up to 475 L, and the

addition of up to 6 wt% EA significantly enhanced the DHCA recovery. Other

compounds in propolis were also easily extracted under this condition, and the

purity of DHCA decreased as more ethyl acetate was added. Experimental data

revealed that the amount of DHCA increased with temperature at pressures from

207 to 276 bar.



2



Green Fluids Extraction and Purification of Bioactive Compounds…



1. CO2 Cylinder



8. Extractor



2-1~2-4. Pressure gaugea



9-1~9-2. Back pressure regulator



3. Gas dryer



10. Micro-metering valve



4. High-pressure pump



11. Absorber



5-1~5-2. Circulator



12. Float flow meter



6-1~6-3. Metering valve



13. Wet gas meter



7. Heat exchanger



14-1~14-5. Thermocouple



77



Fig. 2.3 Schematic diagram of SC-CO2 extraction of propolis (Reprinted from Ref. [14]. With

kind permission of © Elsevier)



2.3.2



Response Surface Methodology (RSM): Designed

Supercritical Carbon Dioxide (SC-CO2) Extractions



Following study of the effects of temperature and cosolvent addition on a few preliminary SC-CO2 extractions, two-factor central composite response surface methodology (RSM) software (Stat-Ease, USA) was adopted to study the effect of the

operating conditions of SC-CO2 extractions on the purity of the DHCA in the

extracts as well as to search for the optimum conditions in this procedure.

The extraction temperature and the addition of the cosolvent were selected as two



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