Comparison of extraction of patchouli (Pogostemon cablin) essential oil with supercritical CO2 and by steam distillation

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The Journal of Supercritical Fluids

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s u p f l u

Review

Comparison of extraction of patchouli (Pogostemon cablin) essential oil with supercritical CO ₂ and by steam distillation

A. Donelian

^a,∗

, L.H.C. Carlson

^b

, T.J. Lopes

^a

, R.A.F. Machado

^a

aLaboratório de Controle de Processos, Departamento de Engenharia Química e Engenharia de Alimentos, Universidade Federal de Santa Catarina, caixa postal 476 CEP: 88010-970, Florianópolis, SC, Brazil

bCoordenac¸ão de Engenharia de Alimentos/CCAA/UNOCHAPECÓ, Av. Sen. Attílio Fontana, 591-E, Bairro Efapi, caixa postal 747, CEP: 89809-000. Chapecó, SC, Brazil

a r t i c l e i n f o

Article history:

Received 12 September 2007

Received in revised form 7 September 2008 Accepted 10 September 2008

Keywords:

Supercritical extraction Patchouli

Essential oil Carbon dioxide

a b s t r a c t

Patchouli essential oil is an important raw material for the perfume and cosmetics industries, besides being

used as a natural additive for food flavoring. Patchoulol and␣-patchoulene are important compounds

of patchouli essential oil, and their concentrations are directly proportional to the quality of the oil.

Nowadays, the usual method employed to obtain patchouli essential oil is steam distillation; however, this causes thermal degradation of some oil compounds. In this study patchouli essential oil was extracted

with supercritical carbon dioxide (scCO₂) under different conditions of pressure (8.5 and 14 MPa) and

temperature (40 and 50^◦C) and also by steam distillation to compare the extraction methods. It was

demonstrated that the extraction with supercritical carbon dioxide provided a higher yield and a better quality of patchouli essential oil.

© 2008 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . 15

2. Experimental . . . 16

2.1. Material . . . 16

2.2. Supercritical equipment . . . 16

2.3. Supercritical extraction procedure . . . 17

2.4. Steam distillation equipment and procedure . . . 17

2.5. Chromatographic analysis . . . 17

3. Results and discussion . . . 17

4. Conclusions . . . 19

References . . . 19

1. Introduction

Patchouli oil is obtained from the leaves ofPogostemon cablin (patchouli), a plant of the Lamiaceae family, originating from Malaysia and India[1]. It is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [2,3,4]. The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products[5,6]. It is also on the FDA’s (Food and Drug

∗ Corresponding author.

E-mail address:adriana@enq.ufsc.br(A. Donelian).

Administration) list of substances approved for human consump- tion, in section 172.510, as a natural additive for food flavoring[7].

Moreover, the plant (P. cablin) is widely used in traditional Chi- nese medicine as it offers various types of pharmacological activity according to the composition of the oil[1,8].

The composition of patchouli oil is unique and complex because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds[5]. The sesquiterpene patchoulol is the major constituent and is the pri- mary component responsible for the typical patchouli aroma. This essential oil is also characterized by a large number of other sesquiterpene hydrocarbons such as ␣-/␤-/␥- patchoulenes,␣- guaiene, seychellene, and␣-himachalene. Although␣-patchoulene is found in small amounts, it is an important constituent of 0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.supflu.2008.09.020

patchouli oil because, together with patchoulol, it also determines the aroma of the oil. Also, it is believed that the antifungal activ- ity of the essential oil is closely related with these two compounds [2,5,8]. Thus, the greater the concentration of these compounds in the essential oil, the better the quality and the higher the commer- cial value[9].

Therefore, as the commercial value of patchouli essential oil is directly correlated with its qualitative and quantitative composi- tion, which varies according to the cultivation region and extraction technique[1,2], an improved process for its extraction would be of industrial interest. It should be noted that patchouli plants are the only commercial source of patchoulol and that cost-effective syn- thetic routes for enantiomerically pure patchoulol have yet to be developed[5].

Nowadays, patchouli essential oil is traditionally obtained by steam distillation[1,2,5]. This procedure, performed at a high tem- perature, can cause the degradation of thermally labile compounds resulting in the formation of undesirable compounds[10]. In this regard, extraction of essential oils using supercritical carbon diox- ide (scCO₂) has been the subject of considerable interest, mainly for the extraction of natural products. Carbon dioxide has sev- eral unique characteristics and physico-chemical properties, since it is non-toxic and inert and has low critical pressure (7.38 MPa) and temperature (31.1^◦C). Compared with conventional extraction methods, extraction with scCO2 has many advantages including more selective extracts without thermal degradation and which are solvent-free, thus providing an oil of superior quality[11,12].

The selectivity of carbon dioxide in relation to the essential oil can be adjusted by changing the temperature and pressure condi- tions, leading to oils with different compositions. Carlson et al.[12]

observed that the best condition for the extraction of lemongrass essential oil was 12 MPa and 40^◦C.

The objective of this study was to compare the variations in the yield and chemical composition of patchouli essential oil obtained under different conditions (pressure and temperature) of supercrit- ical extraction with CO₂and by steam distillation. No information could be found in the literature regarding the use of scCO₂ for patchouli essential oil extraction.

2. Experimental 2.1. Material

Patchouli plants [P. cablin (Blanco) Benth] were collected in November 2002 from “Colônia Penal Agrícola” (Palhoc¸a, SC, Brazil).

For the extraction, the leaves were collected manually from the plants and all of them were from the same lot.

Patchouli leaves were dried in an oven with air circulation (Model TE – 394/2, TECNAL, Brazil) for 1440 min at 30^◦C and 180 min at 35^◦C. These temperatures were selected because they have previously been used in the drying of patchouli leaves for essential oil extraction[13]. The dried leaves were ground with a knife grinder (Model MA – 580, Marconi, Brazil) and, in the case of the supercritical extractions, were then sieved (mesh 30) in order to standardize the size of the particles.

2.2. Supercritical equipment

The extractions with scCO₂ were performed in a pilot unit schematically represented inFig. 1.

A gas booster (3) (Model DLE 15-1, MAXPRO Technologies, Ger- many) received liquid CO₂(99% purity, White Martins, Brazil) from a cylinder (1) and pressurized a jacketed surge tank (6) (Labsolda, UFSC, Brazil, 4.6×10⁻³m³volume) which in turn provided gas to a jacketed extraction vessel (9) (Labsolda, UFSC, Brazil, 1×10⁻³m³

Fig. 1.Experimental unit of supercritical fluid process: (1) CO2cylinder; (2, 4, 8, 14) flow control valves; (3) gas booster; (5 and 10) pressure transducers; (6) jacketed surge tank; (7) pneumatic control valve; (9) jacketed extraction vessel; (11) forward pressure regulator; (12) manometer; (13) separation vessel; (15) micrometer valve;

(16) flow meter; (17 and 18) thermostatic water baths.

volume and 0.55 m height). The jacketed surge tank was placed between the gas booster and the extraction vessel in order to avoid potential pressure overshoots allowing a better pressure control.

The temperatures of the surge tank and extraction vessel were con- trolled by a thermostatic water bath (18) (Model MQBTC 99-20, Microquímica, Brazil).

The extraction pressure was maintained by the gas booster, monitored by a pressure transducer (10) (Model RTP12/BE53R, AEP, Italy) and controlled by a pneumatic control valve (7) (Model: 807, Badger Meter, USA). The samples were collected at different time intervals in a separation vessel (13) at a pressure of 2.4 MPa and temperature of 34^◦C, allowing the separation of the oil by changing the CO2phase. The temperature was maintained by a thermostatic water bath (17) and the pressure by a forward pressure regulator

valve (11) (MTR Model 200-70, Brazil). The solvent flow was con- trolled manually through a micrometer valve (15) (Model SS-21RS4, Swagelok, Ohio, USA) and was measured at the exit of the separator by a flow meter (16) (Model PV005LPMOCC, Key Instruments, USA) under ambient conditions.

2.3. Supercritical extraction procedure

To perform the tests the sieved patchouli plant material was divided into equal portions of 0.15 kg and four extractions were performed. Extractions were carried out at temperatures of 40 and 50^◦C, pressures of 8.5 and 14 MPa, and CO2 flow rate of 6.0×10⁻³kg/min based on the density of the CO₂exiting the sep- arator under ambient conditions. These temperatures were chosen based in the rate commonly used in the supercritical extraction of essential oils[14]and the flow rate was defined as being close to the average CO₂flow rate of 4.7×10⁻³kg/min used by Carlson et al.[12]in the supercritical extraction of lemongrass essential oil.

The influence of pressure and temperature on the patchouli extracted and chemical composition of the patchouli essential oil were evaluated using a two-level factorial design.

Each supercritical extraction experiment was carried out for a period of 340 min. The extract samples were collected in the separation vessel (13) at predefined time intervals and weighed immediately. The time intervals were shorter at the beginning of the extraction and they increased gradually until the end of the extraction. The extracts collected during the experiment were mixed at the end and submitted to chromatographic analysis.

2.4. Steam distillation equipment and procedure

The steam distillation was carried out with approximately 0.1 kg of dry and ground patchouli plant material. The sieved leaves were placed on a perforated plate (3), which was located some centime- ters away from the extractor bottom (4). The extractor was then filled with water up to the plate and heated by a hot-plate (2) pro- ducing water vapor. After passing over the patchouli the vapor was condensed in a cooling system (5), collected in a funnel (6) together with the oil and then separated. The separated oil was collected in an Erlenmeyer flask (7) and weighed immediately after collection.

The extraction experiment was carried out for a period of 120 min, after which time the amount of essential oil collected in the funnel (6) did not increase.

2.5. Chromatographic analysis

Analysis of the samples obtained under different conditions of supercritical CO2 and by steam distillation was performed by Embrapa (Rio de Janeiro, RJ, Brazil).

The separation of essential oil compounds was performed on a Hewlett-Packard gas chromatograph with flame ionization detec- tor (Model HP 5890 series II, USA) using a fused-silica capillary column HP5-MS (25 m length×0.25 mm internal diameter and 0.33␮m film thickness). The flame ionization detector was main- tained at 280^◦C and the injector temperature was 250^◦C. The oven temperature was programmed to increase from 60 to 240^◦C at a rate of 3^◦C/min. The carrier gas was helium at a flow rate of 1.0 mL/min, and the sample volume injected was 0.03␮L of the pure oil with a split rate of 1:100.

Identification of essential oil compounds was based on com- parison of the mass spectra obtained in the gas chromatography with those obtained from the GC–MS library[15]. The chromatogra- phy was performed on a gas chromatograph (Model Agilent 6890), using a fused-silica capillary column HP5-MS, coupled to a selec- tive mass detector (Model 5973N). The injector temperature was

Fig. 2.Extraction curves obtained under different conditions of pressure and tem- perature.

250^◦C, the transfer line temperature 260^◦C, the ion source tem- perature 230^◦C and the ionization mode was electron impact at 70 eV. The oven temperature was programmed to increase from 60 to 240^◦C at a rate of 3^◦C/min. The carrier gas was helium at a flow rate of 1.0×10⁻³L/min and the sample volume injected was 0.03␮L of the pure oil with a split rate of 1:100.

3. Results and discussion

Fig. 2shows the supercritical extraction curves (percentage yield relative to dried patchouli leaves) obtained under different condi- tions of pressure and temperature.

InFig. 2it can be observed that the curves obtained at a pressure of 14 MPa indicate two main stages. The first, known as the stage of constant extract rate, is linear and refers to the stage in which the oil accessible through the cell rupture by grinding is extracted easily by the solvent. After a certain time, which depends on the extraction conditions (e.g. 100 min at 14 MPa and 40^◦C), there is a transition period, in which the extraction decreases rapidly and then continues at a much slower rate, when the oil is extracted from inside the vacuole (second stage). This kind of curve is typical of processes where the solid has a high initial concentration.

However, in extractions at a pressure of 8.5 MPa, the curves did not show the same behavior. This is because both the decrease in pressure and the increase in temperature promoted reductions in the density of the scCO2, as can be observed inTable 1, reducing the solubilization of the oil in the solvent (scCO₂).

In order to analyze statistically the influence of the pressure and temperature on the patchouli essential oil yield at the end of the extraction, the data were organized as shown inTable 2. It can be

Table 1

Patchouli essential oil yield and CO2density under different conditions of supercrit- ical extraction and by steam distillation.

Extraction with scCO2

Pressure (MPa) Temperature (^◦C) Density (kg/m³) Yield (%)

8.5 40 3.57×10⁻⁴ 1.38

8.5 50 2.48×10⁻⁴ 0.44

14 40 7.64×10⁻⁴ 5.07

14 50 6.74×10⁻⁴ 3.92

Steam distillation 1.50

Table 2

Effect of pressure and temperature on patchouli essential oil extraction yield.

Effect^* SEM p −95.0% +95.0%

Mean 2.70 0.0525 0.012366 2.03 3.37

Temperature −1.04 0.1050 0.063753 −2.38 0.29

Pressure 3.58 0.1050 0.018640 2.25 4.92

SEM = Standard error of the mean.

*Estimated values of the effect.

observed, through theplevel value, that only the pressure was sig- nificant at the 95.0% level, since thepvalue for the pressure is less than 0.05. Therefore, an increase in the pressure of the sys- tem can lead to a significant increase in the patchouli essential oil yield during the extraction, while the temperature does not exert a significant influence. Thus, within the conditions studied, the pressure of 14 MPa and temperature of 40^◦C provided the best con- ditions for the extraction of patchouli essential oil in terms of yield.

According to Heath[16]and Moyler[17], the fraction of patchouli oil obtained by steam distillation was between 1.5% and 3.0%, which is in agreement with the value obtained experimentally which was 1.5%. However, the extraction with scCO₂at 14 MPa and 40^◦C pro- vided a much higher yield of 5.07% (no papers or scientific studies relating to the extraction of patchouli essential oil with scCO2could be found for comparison).

Increase in the pressure led to a higher density of the scCO₂ inducing a greater solubilization and, consequently, an increase in the extraction yield, however, with a tendency to reduce the selec- tivity due to the extraction of undesirable components in terms of the oil quality, such as cuticular waxes. Thus, given that the indus- trial interest is mainly concerned with obtaining a product with a greater concentration of active principles Santos[18], the quan- tity of an essential oil is generally evaluated through an analysis of its principal components. In the case of patchouli essential oil, this quality is evaluated from the concentrations of patchoulol, which

is the principal component of the oil, and␣-patchoulene, which is the second most important component.

Thus, in order to evaluate the quality of the patchouli essential oil obtained under the different extraction conditions, chromato- graphic analysis of the oil obtained under each set of conditions was carried out in order to identify the qualitative and quanti- tative composition.Table 3shows the comparative composition of the patchouli essential oil obtained under the different condi- tions.

It can be observed in Table 3 that patchouli essential oil is comprised of a mixture of chemical compounds, most of them in small quantities. The compounds present in higher quantities are the same in all patchouli essential oils: patchoulol,␦-guaiene,␣- guaiene,␣-patchoulene and␤-caryophyllene. These compounds are the same as those found by Betts[6]in the analysis of a commer- cial sample of patchouli essential oil (Rivendell, Bunbury, Western Australia). The commercial sample had 28.5% of patchoulol, 12.0%

of␦-guaiene, 10.4% of␣-guaiene, 6.9% of seychellene, 6.4% of␣- patchoulene, 4.7% of caryophyllene and 1.7% of␤-patchoulene.

Furthermore, on analyzing Table 3 it can be observed that although the increase in pressure promoted an increase in yield, this increase was not associated with a reduction in selectivity. This can be highlighted by the fact that at 40^◦C the increase in pressure from 8.5 to 14 MPa led to an increase in the patchoulol concentration from 14.90% to 31.39%, while the total quantity of the compo- nents identified remained almost the same (80.00% for 8.5 MPa and 78.02% for 14 MPa). This result indicates that the greater yield obtained at high pressure is not exclusively due to the extraction of undesirable components such as cuticular waxes. The results could be further improved with an extraction treatment using scCO₂to remove the waxes, which are normally extracted with the oil. How- ever, in this study this type of treatment was not carried out since the main aim was to assess the viability of the supercritical extrac- tion of patchouli essential oil, in view of the fact that no data are available on this subject in the literature.

Table 3

Main compounds identified in patchouli essential oil.

Compounds Comparative chemical composition (%)

8.5 MPa/40^◦C 8.5 MPa/50^◦C 14 MPa/40^◦C 14 MPa/50^◦C Steam distillation

1-Octen-3-ol 0.12 0.18 0.79 0.08 –

Limonene 0.78 0.40 0.08 0.07 –

Linalool 0.07 0.09 – 0.02 –

␦-Elemene 0.26 0.22 0.17 0.16 –

␤-Patchoulene 2.89 3.26 1.72 1.62 2.6

␤-Elemene 0.99 0.65 0.52 0.50 1.8

␤-Caryophyllene 5.67 5.85 3.29 3.13 4.8

␥-Elemene 0.03 – – 0.03 –

␣-Guaiene 23.05 24.16 14.09 13.38 20.0

␣-Himachalene 0.16 0.17 0.11 0.10 0.9

␣-Patchoulene 5.73 6.21 4.80 4.59 5.8

Seychellene 2.14 2.31 1.83 1.72 3.3

9-Epi-caryophyllene 0.68 0.69 0.48 0.45 0.7

cis-␤-Guaiene 0.08 0.09 0.06 0.06 –

Ledene 0.39 0.41 0.03 0.28 –

␣-Selinene 0.21 0.24 0.49 0.46 3.9

␦-Guaiene 20.75 21.16 16.79 15.55 23.3

␤-Curcumene 0.08 0.10 0.08 0.09 –

7-Epi-␣-selinene 0.19 0.20 0.18 0.17 –

Longicanfenolene 0.41 0.37 0.61 0.62 –

Caryophyllene oxide 0.19 0.16 0.15 0.16 –

Globulel 0.16 0.15 0.24 0.30 –

Epi-␣-cadinel 0.07 – 0.12 0.12 –

Patchoulol 14.90 12.93 31.39 32.23 19.4

␦-Patchoulene – – – – 8.0

␦-himachalene – – – – 0.6

Ftalete – – – – 1.7

Total 80.00 80.00 78.02 75.89 96.8

Table 4

Effect of pressure and temperature on patchoulol concentration.

Effect^* SEM p −95.0% +95.0%

Mean 22.84 0.7275 0.020273 13.59 32.08

Temperature −0.51 1.4550 0.783428 −19.00 17.97

Pressure 17.84 1.4550 0.051793 −0.64 36.33

SEM = Standard error of the mean.

*Estimated values of the effect.

It can be seen inTable 3that the concentrations of patchoulol in the patchouli oil obtained by steam distillation (19.4%) was lower in relation to the oil obtained in the supercritical extractions with a pressure of 14 MPa (31.39% at 40^◦C and 32.23% at 50^◦C), and higher than that obtained with scCO2 at 8.5 MPa (14.90% at 40^◦C and 12.93% at 50^◦C). For the concentrations of␣-patchoulene, essential oil obtained by steam distillation presented a higher concentra- tion (5.8%) in relation to the oil obtained through supercritical extraction at 14 MPa (4.80% at 40^◦C and 4.59% at 50^◦C) and a con- centration close to that of the oil obtained at 8.5 MPa (5.73% at 40^◦C and 6.21% at 50^◦C).

Moreover, the total number of compounds present in the patchouli essential oil (identified and non-identified) obtained by steam distillation was 18, which is lower than the number found in the oils obtained by supercritical extraction (56, 45, 61, and 73 at 8.5 MPa/40^◦C, 8.5 MPa/50^◦C, 14 MPa/40^◦C, and 14 MPa/50^◦C).

These differences in whole the number of compounds and their concentrations in the oils obtained using the two extraction meth- ods may be attributed to the degradation of some components through the use of a high temperature in the steam distillation (approximately 100^◦C).

Nevertheless, it was verified that the quality of oil obtained by steam distillation is very similar to that obtained by supercritical extraction with CO2at the lower pressure (8.5 MPa). However, this quality is lower when compared to the oil obtained with scCO₂ at the higher pressure (14 MPa). Therefore, it was verified that, depending on the pressure condition of scCO2, it is possible to obtain an essential oil of better quality and in higher yield than that obtained by steam distillation.

A statistical analysis was carried out to evaluate the influence of pressure and temperature on the composition of the oils obtained by supercritical extraction. It was found that the pressure has a greater effect than temperature on the patchoulol concentration (Table 4), even though the statistical indices showed a slight ten- dency not to be significant at the 95.0% level. The estimated effect of pressure in relation to the patchoulol concentration is approx- imately 35 times higher than that of temperature. FromTable 4, it can also be noted that the higher pressure tended to promote an increase in the patchoulol concentration. In relation to the con- centration of␣-patchoulene,Table 5shows the statistical analysis of the estimated effect of each factor. It can be observed through the estimated value for the effect that the pressure continues to have the greatest influence on the extraction, although it acts in the opposite way for this component, since increases in pressure tend to lead to a decrease in␣-patchoulene concentration.

Table 5

Effect of pressure and temperature on␣-patchoulene concentration.

Effect^* SEM p −95.0% +95.0%

Mean 5.33 0.1725 0.020587 3.14 7.52

Temperature 0.13 0.3450 0.762549 −4.25 4.52

Pressure −1.27 0.3450 0.168233 −5.66 3.11

SEM = Standard error of the mean.

*Estimated values of the effect.

Therefore, throughTables 4 and 5, it can be noted that the pres- sure is a predominant factor in the variation in the concentration of the principal components of the patchouli essential oil, and the influence of temperature can be neglected. Moreover, on analyzing the estimated values for the effect of pressure on the concentrations of patchoulol and␣-patchoulene, it can be verified that although this factor acts in a different way for each component, its effect is the most significant in relation to the patchoulol concentration, with increases in pressure leading to a patchouli essential oil richer in patchoulol.

As the quality of patchouli essential oil is directly related to the concentration of patchoulol and␣-patchoulene, supercritical extractions with higher pressure and low temperature besides pro- viding an oil of higher yield also provide an oil of better quality.

Thus, in scCO₂extraction, the pressure and temperature conditions which give a higher yield will also provide an oil of better quality.

4. Conclusions

The extraction of patchouli essential oil with scCO₂at 14 MPa and 40^◦C gave the best yield (5.07%), which was higher than that of steam distillation (of 1.50%).

In relation to the essential oil composition, only the pressure had a significant effect on the composition, with a higher pressure leading to a greater concentration of patchoulol, the main compo- nent of the oil. However, regardless of the operating conditions, the compounds present in greatest quantities in all of the patchouli essential oils were the same: patchoulol,␦-guaiene,␣-guaiene,

␣-patchoulene and ␤-caryophyllene. Highest concentrations of patchoulol were obtained in the extractions with supercritical CO2 at 14 MPa, showing that the quality of the patchouli essen- tial oil improved with the increase in the yield. Although the

␣-patchoulene concentration decreased under these conditions, the increase in the patchoulol concentration was of greater signif- icance.

Therefore, for the extraction of patchouli essential oil supercrit- ical carbon dioxide shows better results in terms of yield and oil quality than steam distillation, besides offering the advantage of not promoting the decomposition of possibly thermolabile com- pounds. The best extraction conditions among those tested were 14 MPa and 40^◦C, Future studies will be aimed at defining the optimum operating conditions for the supercritical extraction of patchouli essential oil.

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