RECOVERY OF BETULINIC ACID FROM PLANE TREE BARK (Platanus acerifolia L.)

1Natac Biotech, Parque Científico de Madrid, C/ Faraday, 7, 28049, Madrid, Spain 2Departamento de Producción y Caracterización de Nuevos Alimentos. Instituto de Investigación en Ciencias de la Alimentación

(CIAL) CEI UAM+CSIC, C/ Nicolás Cabrera 9, 28049, Madrid, Spain

J.M. Pinilla1, A. López-Padilla2, G. Vicente2, J.C. Quintela1, G. Reglero2, T. Fornari2

INTRODUCTION

Betulinic acid (BA) (3β, hydroxy-lup-20(29)-en-28-oic acid) is a triterpenic acid which can be isolated from various botanical sources including clove, rosemary, java tea, and

the bark of several betula species (birch trees) [1]. BA as well as its derivatives, have demonstrated a wide range of biological activities, including anti HIV-1 activity, anti-

inflammatory activity, antimalarial activity, anticancer and apoptotic activity [2, 3].

The presence of BA at concentrations up to 3 % (30 mg/g) in the external dried bark of plane (Platanus acerifolia L.) tree was previously reported [1].

In this work different advanced extraction techniques, such as ultrasounds assisted extraction (UAE), pressurized liquid extraction (PLE) and supercritical fluid extraction

(SFE) were studied and compared with conventional solid-liquid extraction (SLE), with the target of recovering BA from the bark of Platanus acerifolia L. Different GRAS

(General Recognized as Safe) solvents were utilized (ethanol, ethyl acetate and SCCO2) and different process conditions were investigated.

RESULTS

EXTRACTION METHODS

CONCLUSIONS

1. Plane tree bark extract with high concentration of BA was obtained by ethanol UAE, followed by a simple fractionation step using water. This approach produced an extract with 46.21 % mass of BA and 2.7 % yield. Furthermore, ethyl acetate UAE can produce almost a two fold increase of extraction yield (5.31 %) with ca. 28 % mass of BA in the extract.

2. The effect of increasing temperature is producing higher yields but lower BA concentrations in the extract (Fig. 1). This conclusion holds for both ethanol and ethyl acetate solvents.

3. The highest BA recovery related with the ethanol consumption was obtained using UAE and SFE (2.4 and 3.0-5.7 mg BA / ml ethanol, respectively). 4. The preliminary SFE accomplished in this work permit to presume that the use of ethanol as CO2 cosolvent has the most significant effect on the extraction of BA from

plane tree bark. In comparison with SLE, UAE and PLE, 20 % ethanol cosolvent resulted in high yield (4.34 %). Good concentration of BA in the extract (18.30 % mass) and almost one third of ethanol consumption. Further investigation to optimize SFE conditions and attain higher BA recovery is necessary.

P. Acerifolia L. Bark dp= 0.5mm

app = 381.7 kg/m3

Moisture = 9.5%

REFERENCES [1] S. Jäger, H. Trojan, T. Kopp, M.N. Laszczyk, A. Scheffler, Molecules, 2009, 14, 2016-2031. [2] P. Yogeeswari P, D. Sriram, Curr. Med. Chem., 2005. 12, 657-666. [3] S. Fulda, Int. J. Mol. Sci., 2008, 9,1096-1107.

AKNOWLEDGMENTS This work has been supported by project ALIBIRD-S2009/AGR-1469 from Comunidad Autónoma de

Madrid. A. López-Padilla thanks to COLCIENCIAS (568-2012) and Medellin Mayor’s office

(Sapiencia/Enlaza Mundos Program, 2013) for his PhD fellowship.

Figure 1. Effect of temperature on the extraction of BA from Platanus acerifolia bark: % mass BA in the extract ( ) , extraction yield () and BA recovery ( ). SLE: 45C; PLE: 100, 150 and 200C. (a) Ethanol; (b) ethyl acetate solvent.

0

5

10

15

20

25

30

35

45 100 150 200

temperature ( C)

(a)

0

5

10

15

20

25

30

35

45 100 150 200

temperature ( C)

(b)

Figure 2. Comparison between the extraction methods using liquid ethanol (SLE, UAE and PLE) and SFE with 0, 10 and 20 % ethanol cosolvent (a) BA recovery; (b) BA concentration; (c) ethanol consumption.

SLE

Ratio= 1:10 Solvents= EtOH; Ethyl Acetate

T(C)= 45 t(h)= 1.5

PLE

Ratio= 1:10 Solvents= EtOH; Ethyl

Acetate T( C)= 100;150;200

t(min)= 15

UAE

Ratio= 1:5 Solvents= EtOH; Ethyl Acetate

Amplitude: 70% T( C)= 45 t(min)= 15

0

3

6

9

12

15

SLEethanol(45°C)

PLEethanol(100°C)

UAEethanol(45°C)

SFE-0%(50 MPa,

40°C)

SFE-10%(30 MPa,

40°C)

SFE-20%(30 MPa,

40°C)

BA

re

cov

ery

(m

g /

g b

ark

) (a)

0

10

20

30

40

50

SLEethanol(45°C)

PLEethanol(100°C)

UAEethanol(45°C)

SFE-0%(50 MPa,

40°C)

SFE-10%(30 MPa,

40°C)

SFE-20%(30 MPa,

40°C)

% m

ass

BA

(b)

0

3

6

9

12

15

SLEethanol(45°C)

PLEethanol(100°C)

UAEethanol(45°C)

SFE-0%(50 MPa,

40°C)

SFE-10%(30 MPa,

40°C)

SFE-20%(30 MPa,

40°C)

ml

eth

an

ol /

g B

A

(c)

SFE

Mass (kg)= 0.57 P(MPa)= 25;30;50

T(C)= 40 CO2 flow rate= 50 g/min

Cosolvent= EtOH (10; 20%) t(h)= 1.5; 2.5; 4.0

FRACTIONATION OF ETHANOLIC EXTRACTS

EtOH extraction EtOH extraction

reduction 1/3 of the initial volume at low pressure reduction 1/3 of the initial volume at low pressure

addition 1/3 of H2O and store 30 min at room temperature. addition 1/3 of H2O and store 30 min at room temperature.

after filtration solid phase was freeze-dried after filtration solid phase was freeze-dried

liquid phase was evaporated at low pressure and freeze-dried liquid phase was evaporated at low pressure and freeze-dried

product (BA concentrate) product (BA concentrate)

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5

6

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