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Dual extraction of essential oil and podophyllotoxin from Juniperus virginiana
Industrial Crops and Products 30 (2009) 276–280
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Dual extraction of essential oil and podophyllotoxin from Juniperus virginiana Archana J. Gawde a , Charles L. Cantrell b , Valtcho D. Zheljazkov a,c,∗ a
Mississippi State University, Department of Plant and Soil Sciences, Mississippi State, MS 39762, United States Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 8048, University, MS 38677, United States c Mississippi State University, North Mississippi Research and Extension Center, 5421 Highway 145 South, Verona, MS 38879, United States b
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 8 May 2009 Accepted 14 May 2009 Keywords: Podophyllotoxin Essential oil Dual extraction Juniperus virginiana Cupressaceae
a b s t r a c t The leaves (needles) of eastern red cedar (Juniperus virginiana L.) contain two important natural products: essential oil and podophyllotoxin. The hypothesis of this study was that it may be possible to extract both essential oil and podophyllotoxin from the leaves of the tree, by using a dual extraction method. Podophyllotoxin was obtained from the leaves following steam distillation of the leaves to produce the essential oil, indicating that steam distillation did not degrade podophyllotoxin. Furthermore, a product with 6% purity podophyllotoxin was obtained from the steam-distilled plant material, demonstrating the possibility for the establishment of an industrially economic protocol for dual extraction of these two natural products. Our study demonstrated that J. virginiana leaves, currently a waste-product from the timber industry, could be sequentially extracted for essential oil and podophyllotoxin and utilized as a by-product instead. We also found that the J. virginiana heartwood (a traditional source for cedarwood essential oil) does not contain podophyllotoxin. This is the first study to report both podophyllotoxin and essential oil in J. virginiana, and the first report on the dual extraction of these two natural products from the same biomass samples. Published by Elsevier B.V.
1. Introduction J. virginiana L. (Family Cupressaceae) commonly called eastern red cedar is a widely distributed species in the USA and parts of Canada. J. virginiana heartwood is well-known for its use of durable, termite resistant and insect resistant heartwood (redwood). The heartwood is also used for commercial production of essential oil, commonly termed cedarwood oil. J. virginiana leaves also contain podophyllotoxin, a precursor lignan for anticancer compounds (Hartwell et al., 1953). Pharmaceutical companies obtain podophyllotoxin primarily from Indian mayapple [Podophyllum emodi Wall. (synonym Podophyllum hexandrum Royle)], now considered endangered. American mayapple (Podophyllum peltatum L.), a native plant in North America, has been suggested as an alternative source for podophyllotoxin (Meijer, 1974) but was never introduced as crop because of various challenges. Podophyllotoxin concentration in the leaves of American mayapple is generally seven times higher than in J. virginiana leaves. However, a recent study demonstrated that podophyllotoxin concentration in American mayapple in its natural habitats vary significantly, with some mayapple colonies lacking any podophyllotoxin (Zheljazkov et al., 2009). In addition, J. vir-
∗ Corresponding author at: Mississippi State University, North Mississippi Research and Extension Center, 5421 Highway 145 South, Verona, MS 38879, United States. Tel.: +1 662 566 2201; fax: +1 662 566 2257. E-mail address: [email protected] (V.D. Zheljazkov). 0926-6690/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.indcrop.2009.05.005
giniana can produce much more biomass than American mayapple. Hence, J. virginiana may be a more economically and environmentally sustainable source for the compound because of (1) much higher biomass production; (2) wider distribution; (3) less complicated cultivation techniques and wider adaptability (if grown as a crop) and (4) possibility to be used year round, being an evergreen. J. virginiana heartwood oil has been studied extensively for its oil content, composition and yields (Coleman and Lawrence, 1997; Payne et al., 1999; Eller and King, 2000; Dunford et al., 2007). However, few studies have been reported on the composition and yields of essential oil from J. virginiana leaves (Semen and Hiziroglu, 2005; Dunford et al., 2007), a by-product of the timber industry. There are no reports investigating both essential oil and podophyllotoxin from the leaves of J. virginiana. We hypothesized that dual extraction of essential oil and podophyllotoxin from the J. virginiana leaves may be possible, simultaneously or sequentially. Furthermore, we hypothesized that if podophyllotoxin is not degraded during steam distillation, it may be recoverable either from the steam-distilled plant material or water from distillation. This paper discusses the development of dual extraction procedure for essential oil and podophyllotoxin from J. virginiana leaves.
2. Materials and methods J. virginiana heartwood, sapwood, bark and leaves (needles) were first tested for the presence of podophyllotoxin. Of all these
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presence of podophyllotoxin to see which fraction could be used further as a source of podophyllotoxin. The amount of podophyllotoxin obtained was also estimated to calculate the amount of loss of podophyllotoxin if any. The methodology used in the present experiments has been summarized in Fig. 2. 2.1. Collection, drying, and preparation of plant material The plant material for the experiments was obtained from J. virginiana trees found at the North Mississippi Research and Extension Center in Verona, MS (34◦ 43 22 N and 88◦ 43 22 W). Samples from three different trees were collected and one sample each of the leaves was made for steam distillation. All experiments were conducted in triplicate. The plant material (leaves on branches not ticker than 3 mm) was dried in an oven at 40 ◦ C for 48 h. After that, the plant tissue samples were chopped into small pieces (approximately 2 cm long). 2.2. Distillation
Fig. 1. Schematic drawing of a typical post-distillation setup used in the generation of essential oils.
only leaves showed the presence of podophyllotoxin. This is why we considered leaves in the present study. The method involves the steam distillation of the leaves first to obtain four fractions: essential oil, steam-distilled (residual) plant material, residual water and hydrolat (Fig. 1). The residual water is the water collecting below the steam-distilled plant material; it results from the condensation of some of the hot steam passing through the material. The hydrolat is the water eluted from the distillation apparatus after the separation with the essential oil. Each of these products was tested for
400 g of the dried and chopped plant material was steamdistilled in a 2-L steam distillation unit (Fig. 1) (Furnis et al., 1989) for 90 min, at 100–105 ◦ C and a flow rate of approximately 7.4 mL/min. The amount of oil was expressed (w/w) as essential oil/dried raw material. The essential oil was collected and stored under refrigeration. The residual plant material (post-distillation) was retrieved and dried in an oven at 40 ◦ C for 48 h in paper bags. The volume of the residual water from distillation (Fig. 1) was recorded and was stored under refrigeration to be used for analysis of podophyllotoxin. The water that was drained off during oil accumulation, i.e. ‘hydrolat’ (Fig. 1), was also collected, the volume was measured and analyzed for podophyllotoxin. 2.3. Podophyllooxin extraction Representative samples of 40 mg of dried plant material (steam-distilled and not steam-distilled) were mixed in 0.6 mL of phosphate buffer (pH 7) and mixed on an eppendorf ther-
Fig. 2. Flow-chart of steps used in developing dual extraction method for podophyllotoxin and essential oil from Juniperus virginiana.
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A.J. Gawde et al. / Industrial Crops and Products 30 (2009) 276–280 Table 1 Amount of podophyllotoxin obtained from untreated plant material and extracted fractions of Juniperus virginiana leaves. Sample
Podophyllotoxin (% DW)
Steam-distilled Untreated Residual watera Essential oil Hydrolat
0.218 ± 0.022 0.221 ± 0.014 0.00076 ± 0.00013 Absent Absent
a
Residual water extracted by second method.
momixer for 30 min at 800 rpm. Then, 0.8 mL of ethyl acetate was added to each sample and incubated for another 10 min. The ethyl acetate phase was separated using centrifugation (Savant speed vac. svc 200), collected, and dried under a stream of nitrogen. The residue was re-dissolved in high performance liquid chromatographic (HPLC) grade methanol (1 mL) and 10 L used for HPLC analysis (Canel et al., 2001). In another experiment, the residual water and hydrolat were basified using a phosphate buffer (pH 7) and incubated for 30 min with shaking. One mL of this was transferred to an HPLC vial for analysis of podophyllotoxin. The essential oil samples were diluted to a concentration of 1 mg/mL using methanol and also used for podophyllotoxin analysis. A large scale experiment was conducted with 10 g of plant material, both steam-distilled and untreated, to determine the purity of podophyllotoxin in the ethyl acetate extractables. Samples of 10 g of plant material were basified with 300 mL phosphate buffer (pH 7) and incubated for 30 min with intermittent shaking. Four hundred mL of ethyl acetate was added and incubated for another 10 min with shaking. The ethyl acetate fraction was separated using a glass vacuum filter. This fraction was evaporated to dryness on a Buchi Rotavap R-114 (Buchi Labortechnik AG, Switzerland), equipped with a water bath B-480 (Neslab-RTE-140). The dried residue was weighed and 3 mg of this residue was dissolved in 1 mL of HPLC grade methanol. This was used for HPLC analysis. 2.4. HPLC analysis of podophyllotoxin Extracts of steam-distilled and untreated plant material were analyzed using isocratic HPLC for 20 min with a flow rate of 1 mL/min, followed by a 10 min methanol wash and a reequilibration for 15 min. The instrument consisted of an Agilent 1200 series instrument with a vacuum degasser, quaternary pump, autosampler, diode array detector, and an Agilent Eclipse XDB-C18 4.6 mm × 150 mm, 5 m column. One mL was transferred to an HPLC vial and directly injected for podophyllotoxin analysis. The injection volume was 10 L. All sample injections were analyzed at room temperature. The mobile phase was acetonitrile:water (28:72) 0.025% trifluoroacetic acid (TFA), max = 220 nm. A standard curve was obtained with a podophyllotoxin reference standard (0.01–1 mg/mL). All chemicals used for HPLC analysis were HPLC grade (Fisher Scientific, Hampton, NH). 2.5. Gas chromatography–mass spectrophotometer (GC–MS) analysis of the essential oil Qualitative and quantitative analysis of the leaf essential oil obtained by steam distillation was conducted. GC–MS methods for
analysis and conditions are identical to those previously described (Zheljazkov et al., 2008). Individual concentration gradients for reference standards were prepared for -pinene, myrcene, linalool, iso-safrole, limonene, safrole and bornyl acetate, to obtain a standard curve for each. The oil composition was determined by obtaining individual standard curves for -pinene (R2 = 0.9999), myrcene (R2 = 0.9993), linalool (R2 = 0.9991), (E)-iso-safrole (R2 = 0.9998), (Z)-iso-safrole (R2 = 0.9636), limonene (R2 = 0.9996), safrole (R2 = 0.9995) and bornyl acetate (R2 = 0.9997). 3. Results and discussion 3.1. Analysis of podophyllotoxin As indicated above, J. virginiana heartwood, sapwood, and bark were tested but did not show the presence of podophyllotoxin. Hence, only the leaves (needles) of J. virginiana were used for the present experiment. The amount of podophyllotoxin was estimated in untreated plant material, steam-distilled plant material (residual plant material after the distillation of oil), residual water, hydrolat and essential oil (Table 1). Podophyllotoxin was present in both the steam-distilled plant material and in residual water. However, the amount extracted from the residual water was negligible (0.00076 ± 0.00013% DW). The amount of podophyllotoxin in the steam-distilled plant material was 0.218 ± 0.022% DW, which was approximately equal to the detected in untreated plant material (0.221 ± 0.014% DW). This indicates that podophyllotoxin was not degraded during the distillation process. The hydrolat and the essential oil did not indicate presence of podophyllotoxin. 3.2. Purity and recovery of podophyllotoxin A large scale experiment was done with 10 g of plant material, both steam-distilled and untreated, to determine the purity of podophyllotoxin in ethyl acetate (Table 2). The steam-distilled and untreated plant material yielded an ethyl acetate residue of 0.2515 ± 0.015 mg and 0.296 ± 0.037 mg respectively. A high purity percentage of podophyllotoxin was obtained, i.e. 6.228 ± 0.689% and 5.8012 ± 1.889% in steam-distilled and untreated respectively. The total recovery of podophyllotoxin was not significantly different from its recovery from untreated plant material; with 67.23% and 77.76% in steam-distilled and untreated plant material respectively (Table 2). 3.3. Essential oil analysis A total average yield of oil obtained by steam distillation from J. virginiana leaves was 0.696 ± 0.113 g from 400 g of plant material (0.174%, w/w). The essential oil was further characterized by quantifying the oil components. Since there is no standard essential oil of J. viriginiana leaf, only individual components which were commercially available were quantified on the basis of previous reports (Von Rudloff, 1975; Dunford et al., 2007). Eight essential oil components (-pinene, myrcene, linalool, -(E)-iso-safrole, (Z)-isosafrole, limonene, safrole and bornyl acetate) were quantified using standard GC by obtaining the standard curves for these reference compounds (Table 3).
Table 2 Large scale experiments (10 g) for the extraction of podophyllotoxin from untreated and steam-distilled plant material. Podophyllotoxin (% DW) Steam-distilled Untreated a
0.156 ± 0.065 0.172 ± 0.062
Amount of EtoAc residue (g)
Purity of podophyllotoxin EtoAc (%)
%Recoverya
0.2515 ± 0.015 0.0296 ± 0.037
6.228 ± 0.689 5.801 ± 1.889
72 78
Recovery of a compound is determined by comparing the actual amount present and the amount obtained by extraction on a large scale.
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Fig. 3. GC–MS profile of essential oil obtained from leaves of J. virginiana. (a) -Pinene, (b) myrcene, (c) limonene, (d) linalool, (e) bornyl acetate, (f) safrole, (g) (Z)-iso-safrole, and (h) (E)-iso-safrole.
Safrole was reported as the major active constituent of essential oil of leaves obtained from Texas accessions (Von Rudloff, 1975), whereas that of Ontario had a low concentration of safrole (1.5%). The essential oil obtained in the present study (Fig. 3 and Table 3); shows a high concentration of safrole, i.e. 19.012 ± 3.8086%, closely followed by limonene (18.195 ± 0.6184%). Limonene was reported to be in high amounts in Ontario accessions of J. virginiana leaves with a concentration of 17.8%, whereas those collected from Texas produced only 0.9% of limonene. Another study on essential oil obtained from leaves of plant material collected from Oklahoma, reports the presence of 2.6% limonene (Dunford et al., 2007). In the present study the concentration of other components (-pinene, myrcene, linalool, (E)-iso-safrole, (Z)-iso-safrole and bornyl acetate) was low as compared to safrole and limonene. Previous reports found a variation in the content of these components from accessions collected from Texas, Ontario and Oklahoma, with 10.6% myrcene and 0.9% bornyl acetate being reported from Oklahoma accessions (Von Rudloff, 1975; Dunford et al., 2007), and 1.4% and 0.7% linalool from Texas and Ontario, respectively (Von Rudloff, 1975). The simultaneous extraction of essential oil and podophyllotoxin, without having to lose any podophyllotoxin during the steam distillation process is perhaps the most important finding of this study. J. virginiana is an evergreen and is abundantly available, to an extent that in some states it has been considered invasive (Gold et al., 2005). J. virginiana can be a consistent source of podophyllotoxin and Juniper leaf essential oil. Employing the present method of dual extraction of J. virginiana leaves offers an alternative source use for by-product, from which two commercially viable natural products can be obtained. In addition, we demonstrated that an approximately 6% pure product of podophyllotoxin could be obtained from the steam-distilled red cedar leaves. Although 6% purity is excellent for only a few steps, higher purity may be desirable for commercial
Table 3 Analysis of components of leaf essential oil of Juniperus virginiana. Sr. No.
Compound
1 2 3 4 5 6 7 8
Safrole Limonene -pinene Myrcene Linalool (E)-iso-safrole (Z)-iso-safrole Bornyl acetate
Concentration (%) 19.012 18.195 3.041 1.378 1.137 0.247 0.165 0.01
± ± ± ± ± ± ± ±
3.8086 0.6184 0.4264 0.2128 0.1505 0.1489 0.0566 0.00372
applications. Further research is needed to purify this product by column separations or precipitation experiments. 4. Conclusions This work demonstrated that both essential oil and podophyllotoxin can be extracted from the same biomass samples of J. virginiana leaves. It was found that the process of steam distillation of the essential oil did not degrade podophyllotoxin. Following the distillation of the essential oil, podophyllotoxin could be recovered from both the steam-distilled biomass and from the residual water. It was also found that J. virginiana heartwood (a traditional source for Juniper essential oil) does not contain podophyllotoxin. Acknowledgements Contribution of the Mississippi Agricultural and Forestry Exp. Sta. journal article No J-11568. This study was supported in part by USDA-NRI project “American mayapple and eastern red cedar as domestic sources for the anti-cancer compound podophyllotoxin”, and Mississippi State University/MAFES project “Bioprospecting for anti-cancer compound podophyllotoxin”. Authors thank the MAFES for continuous support. Authors also thank Amber Callahan for assistance with quantitative analysis. References Canel, C., Dayan, F.E., Ganzera, M., Khan, I.A., Rimando, A., Burandt, C., Moraes, M., 2001. High yield of podophyllotoxin from leaves of Podophyllum peltatum by in situ conversion of podophyllotoxin 4-O-b-d-glucopyranoside. Planta Med. 67, 97–99. Coleman, W.M., Lawrence, B.M., 1997. A comparison of selected analytical approaches to the analysis of an essential oil. Flavour Frag. J. 12, 1–8. Dunford, N.T., Hiziroglu, S., Holcomb, R., 2007. Effect of age on the distribution of oil in eastern red cedar tree segments. Bioresour. Technol. 98 (14), 2636– 2640. Eller, F.J., King, J.W., 2000. Supercritical carbon dioxide extraction of cedarwood oil: a study of extraction parameters and oil characteristics. Phytochem. Anal. 11, 226–231. Furnis, B.S., Hannaford, A.J., Smith, P.W.G., Tatchell, A.R., 1989. Vogel’s Textbook of Practical Chemistry, 5th ed. Longman Scientific & Technical, New York, NY, pp. 171–175. Gold, M.A., Godsey, L.D., Cernusca, M.M., 2005. Comparative market analysis of eastern red cedar. Forest Prod. J. 55, 58–65. Hartwell, J.L., Johnson, J.M., Fitzgerald, D.B., Belkin, M., 1953. Podophyllotoxin from Juniperus species; Savinin. J. Am. Chem. Soc. 75 (1), 235–236. Meijer, W., 1974. Podophyllum peltatum – mayapple: a potential new cash crop plant of eastern North America. Econ. Bot. 28 (1), 68–72. Payne, K.W., Wittwear, R., Anderson, S., Eisenbraun, E.J., 1999. Use of a modified abderhalden apparatus for comparing laboratory and industrial methods for
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isolating eastern red cedar essential oil. Forest Prod. J. 49, 90–92. Semen, E., Hiziroglu, S., 2005. Production, yield and derivatives of volatile oils from eastern redcedar (Juniperus virginiana L.). Am. J. Environ. Sci. 1, 133– 138. Von Rudloff, E., 1975. Volatile leaf oil analysis in chemosynthetic studies of North American conifer. Biochem. Syst. Ecol. 2, 131–167.
Zheljazkov, V.D., Cantrell, C.L., Tekwani, B., Khan, S., 2008. Content, composition, and bioactivity of the essential oil of three basil genotypes as a function of harvesting. J. Agric. Food Chem. 56 (2), 380–385. Zheljazkov, V.D., Avula, B., Jones, A.M., Maddox, V., Rowe, D.E., 2009. Lignan and nutrient concentrations in American Mayapple (Podophyllum peltatum L.) in the Eastern United States. HortScience 44 (2), 349–353.
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Dual extraction of essential oil and podophyllotoxin from Juniperus virginiana Archana J. Gawde a , Charles L. Cantrell b , Valtcho D. Zheljazkov a,c,∗ a
Mississippi State University, Department of Plant and Soil Sciences, Mississippi State, MS 39762, United States Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 8048, University, MS 38677, United States c Mississippi State University, North Mississippi Research and Extension Center, 5421 Highway 145 South, Verona, MS 38879, United States b
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 8 May 2009 Accepted 14 May 2009 Keywords: Podophyllotoxin Essential oil Dual extraction Juniperus virginiana Cupressaceae
a b s t r a c t The leaves (needles) of eastern red cedar (Juniperus virginiana L.) contain two important natural products: essential oil and podophyllotoxin. The hypothesis of this study was that it may be possible to extract both essential oil and podophyllotoxin from the leaves of the tree, by using a dual extraction method. Podophyllotoxin was obtained from the leaves following steam distillation of the leaves to produce the essential oil, indicating that steam distillation did not degrade podophyllotoxin. Furthermore, a product with 6% purity podophyllotoxin was obtained from the steam-distilled plant material, demonstrating the possibility for the establishment of an industrially economic protocol for dual extraction of these two natural products. Our study demonstrated that J. virginiana leaves, currently a waste-product from the timber industry, could be sequentially extracted for essential oil and podophyllotoxin and utilized as a by-product instead. We also found that the J. virginiana heartwood (a traditional source for cedarwood essential oil) does not contain podophyllotoxin. This is the first study to report both podophyllotoxin and essential oil in J. virginiana, and the first report on the dual extraction of these two natural products from the same biomass samples. Published by Elsevier B.V.
1. Introduction J. virginiana L. (Family Cupressaceae) commonly called eastern red cedar is a widely distributed species in the USA and parts of Canada. J. virginiana heartwood is well-known for its use of durable, termite resistant and insect resistant heartwood (redwood). The heartwood is also used for commercial production of essential oil, commonly termed cedarwood oil. J. virginiana leaves also contain podophyllotoxin, a precursor lignan for anticancer compounds (Hartwell et al., 1953). Pharmaceutical companies obtain podophyllotoxin primarily from Indian mayapple [Podophyllum emodi Wall. (synonym Podophyllum hexandrum Royle)], now considered endangered. American mayapple (Podophyllum peltatum L.), a native plant in North America, has been suggested as an alternative source for podophyllotoxin (Meijer, 1974) but was never introduced as crop because of various challenges. Podophyllotoxin concentration in the leaves of American mayapple is generally seven times higher than in J. virginiana leaves. However, a recent study demonstrated that podophyllotoxin concentration in American mayapple in its natural habitats vary significantly, with some mayapple colonies lacking any podophyllotoxin (Zheljazkov et al., 2009). In addition, J. vir-
∗ Corresponding author at: Mississippi State University, North Mississippi Research and Extension Center, 5421 Highway 145 South, Verona, MS 38879, United States. Tel.: +1 662 566 2201; fax: +1 662 566 2257. E-mail address: [email protected] (V.D. Zheljazkov). 0926-6690/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.indcrop.2009.05.005
giniana can produce much more biomass than American mayapple. Hence, J. virginiana may be a more economically and environmentally sustainable source for the compound because of (1) much higher biomass production; (2) wider distribution; (3) less complicated cultivation techniques and wider adaptability (if grown as a crop) and (4) possibility to be used year round, being an evergreen. J. virginiana heartwood oil has been studied extensively for its oil content, composition and yields (Coleman and Lawrence, 1997; Payne et al., 1999; Eller and King, 2000; Dunford et al., 2007). However, few studies have been reported on the composition and yields of essential oil from J. virginiana leaves (Semen and Hiziroglu, 2005; Dunford et al., 2007), a by-product of the timber industry. There are no reports investigating both essential oil and podophyllotoxin from the leaves of J. virginiana. We hypothesized that dual extraction of essential oil and podophyllotoxin from the J. virginiana leaves may be possible, simultaneously or sequentially. Furthermore, we hypothesized that if podophyllotoxin is not degraded during steam distillation, it may be recoverable either from the steam-distilled plant material or water from distillation. This paper discusses the development of dual extraction procedure for essential oil and podophyllotoxin from J. virginiana leaves.
2. Materials and methods J. virginiana heartwood, sapwood, bark and leaves (needles) were first tested for the presence of podophyllotoxin. Of all these
A.J. Gawde et al. / Industrial Crops and Products 30 (2009) 276–280
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presence of podophyllotoxin to see which fraction could be used further as a source of podophyllotoxin. The amount of podophyllotoxin obtained was also estimated to calculate the amount of loss of podophyllotoxin if any. The methodology used in the present experiments has been summarized in Fig. 2. 2.1. Collection, drying, and preparation of plant material The plant material for the experiments was obtained from J. virginiana trees found at the North Mississippi Research and Extension Center in Verona, MS (34◦ 43 22 N and 88◦ 43 22 W). Samples from three different trees were collected and one sample each of the leaves was made for steam distillation. All experiments were conducted in triplicate. The plant material (leaves on branches not ticker than 3 mm) was dried in an oven at 40 ◦ C for 48 h. After that, the plant tissue samples were chopped into small pieces (approximately 2 cm long). 2.2. Distillation
Fig. 1. Schematic drawing of a typical post-distillation setup used in the generation of essential oils.
only leaves showed the presence of podophyllotoxin. This is why we considered leaves in the present study. The method involves the steam distillation of the leaves first to obtain four fractions: essential oil, steam-distilled (residual) plant material, residual water and hydrolat (Fig. 1). The residual water is the water collecting below the steam-distilled plant material; it results from the condensation of some of the hot steam passing through the material. The hydrolat is the water eluted from the distillation apparatus after the separation with the essential oil. Each of these products was tested for
400 g of the dried and chopped plant material was steamdistilled in a 2-L steam distillation unit (Fig. 1) (Furnis et al., 1989) for 90 min, at 100–105 ◦ C and a flow rate of approximately 7.4 mL/min. The amount of oil was expressed (w/w) as essential oil/dried raw material. The essential oil was collected and stored under refrigeration. The residual plant material (post-distillation) was retrieved and dried in an oven at 40 ◦ C for 48 h in paper bags. The volume of the residual water from distillation (Fig. 1) was recorded and was stored under refrigeration to be used for analysis of podophyllotoxin. The water that was drained off during oil accumulation, i.e. ‘hydrolat’ (Fig. 1), was also collected, the volume was measured and analyzed for podophyllotoxin. 2.3. Podophyllooxin extraction Representative samples of 40 mg of dried plant material (steam-distilled and not steam-distilled) were mixed in 0.6 mL of phosphate buffer (pH 7) and mixed on an eppendorf ther-
Fig. 2. Flow-chart of steps used in developing dual extraction method for podophyllotoxin and essential oil from Juniperus virginiana.
278
A.J. Gawde et al. / Industrial Crops and Products 30 (2009) 276–280 Table 1 Amount of podophyllotoxin obtained from untreated plant material and extracted fractions of Juniperus virginiana leaves. Sample
Podophyllotoxin (% DW)
Steam-distilled Untreated Residual watera Essential oil Hydrolat
0.218 ± 0.022 0.221 ± 0.014 0.00076 ± 0.00013 Absent Absent
a
Residual water extracted by second method.
momixer for 30 min at 800 rpm. Then, 0.8 mL of ethyl acetate was added to each sample and incubated for another 10 min. The ethyl acetate phase was separated using centrifugation (Savant speed vac. svc 200), collected, and dried under a stream of nitrogen. The residue was re-dissolved in high performance liquid chromatographic (HPLC) grade methanol (1 mL) and 10 L used for HPLC analysis (Canel et al., 2001). In another experiment, the residual water and hydrolat were basified using a phosphate buffer (pH 7) and incubated for 30 min with shaking. One mL of this was transferred to an HPLC vial for analysis of podophyllotoxin. The essential oil samples were diluted to a concentration of 1 mg/mL using methanol and also used for podophyllotoxin analysis. A large scale experiment was conducted with 10 g of plant material, both steam-distilled and untreated, to determine the purity of podophyllotoxin in the ethyl acetate extractables. Samples of 10 g of plant material were basified with 300 mL phosphate buffer (pH 7) and incubated for 30 min with intermittent shaking. Four hundred mL of ethyl acetate was added and incubated for another 10 min with shaking. The ethyl acetate fraction was separated using a glass vacuum filter. This fraction was evaporated to dryness on a Buchi Rotavap R-114 (Buchi Labortechnik AG, Switzerland), equipped with a water bath B-480 (Neslab-RTE-140). The dried residue was weighed and 3 mg of this residue was dissolved in 1 mL of HPLC grade methanol. This was used for HPLC analysis. 2.4. HPLC analysis of podophyllotoxin Extracts of steam-distilled and untreated plant material were analyzed using isocratic HPLC for 20 min with a flow rate of 1 mL/min, followed by a 10 min methanol wash and a reequilibration for 15 min. The instrument consisted of an Agilent 1200 series instrument with a vacuum degasser, quaternary pump, autosampler, diode array detector, and an Agilent Eclipse XDB-C18 4.6 mm × 150 mm, 5 m column. One mL was transferred to an HPLC vial and directly injected for podophyllotoxin analysis. The injection volume was 10 L. All sample injections were analyzed at room temperature. The mobile phase was acetonitrile:water (28:72) 0.025% trifluoroacetic acid (TFA), max = 220 nm. A standard curve was obtained with a podophyllotoxin reference standard (0.01–1 mg/mL). All chemicals used for HPLC analysis were HPLC grade (Fisher Scientific, Hampton, NH). 2.5. Gas chromatography–mass spectrophotometer (GC–MS) analysis of the essential oil Qualitative and quantitative analysis of the leaf essential oil obtained by steam distillation was conducted. GC–MS methods for
analysis and conditions are identical to those previously described (Zheljazkov et al., 2008). Individual concentration gradients for reference standards were prepared for -pinene, myrcene, linalool, iso-safrole, limonene, safrole and bornyl acetate, to obtain a standard curve for each. The oil composition was determined by obtaining individual standard curves for -pinene (R2 = 0.9999), myrcene (R2 = 0.9993), linalool (R2 = 0.9991), (E)-iso-safrole (R2 = 0.9998), (Z)-iso-safrole (R2 = 0.9636), limonene (R2 = 0.9996), safrole (R2 = 0.9995) and bornyl acetate (R2 = 0.9997). 3. Results and discussion 3.1. Analysis of podophyllotoxin As indicated above, J. virginiana heartwood, sapwood, and bark were tested but did not show the presence of podophyllotoxin. Hence, only the leaves (needles) of J. virginiana were used for the present experiment. The amount of podophyllotoxin was estimated in untreated plant material, steam-distilled plant material (residual plant material after the distillation of oil), residual water, hydrolat and essential oil (Table 1). Podophyllotoxin was present in both the steam-distilled plant material and in residual water. However, the amount extracted from the residual water was negligible (0.00076 ± 0.00013% DW). The amount of podophyllotoxin in the steam-distilled plant material was 0.218 ± 0.022% DW, which was approximately equal to the detected in untreated plant material (0.221 ± 0.014% DW). This indicates that podophyllotoxin was not degraded during the distillation process. The hydrolat and the essential oil did not indicate presence of podophyllotoxin. 3.2. Purity and recovery of podophyllotoxin A large scale experiment was done with 10 g of plant material, both steam-distilled and untreated, to determine the purity of podophyllotoxin in ethyl acetate (Table 2). The steam-distilled and untreated plant material yielded an ethyl acetate residue of 0.2515 ± 0.015 mg and 0.296 ± 0.037 mg respectively. A high purity percentage of podophyllotoxin was obtained, i.e. 6.228 ± 0.689% and 5.8012 ± 1.889% in steam-distilled and untreated respectively. The total recovery of podophyllotoxin was not significantly different from its recovery from untreated plant material; with 67.23% and 77.76% in steam-distilled and untreated plant material respectively (Table 2). 3.3. Essential oil analysis A total average yield of oil obtained by steam distillation from J. virginiana leaves was 0.696 ± 0.113 g from 400 g of plant material (0.174%, w/w). The essential oil was further characterized by quantifying the oil components. Since there is no standard essential oil of J. viriginiana leaf, only individual components which were commercially available were quantified on the basis of previous reports (Von Rudloff, 1975; Dunford et al., 2007). Eight essential oil components (-pinene, myrcene, linalool, -(E)-iso-safrole, (Z)-isosafrole, limonene, safrole and bornyl acetate) were quantified using standard GC by obtaining the standard curves for these reference compounds (Table 3).
Table 2 Large scale experiments (10 g) for the extraction of podophyllotoxin from untreated and steam-distilled plant material. Podophyllotoxin (% DW) Steam-distilled Untreated a
0.156 ± 0.065 0.172 ± 0.062
Amount of EtoAc residue (g)
Purity of podophyllotoxin EtoAc (%)
%Recoverya
0.2515 ± 0.015 0.0296 ± 0.037
6.228 ± 0.689 5.801 ± 1.889
72 78
Recovery of a compound is determined by comparing the actual amount present and the amount obtained by extraction on a large scale.
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Fig. 3. GC–MS profile of essential oil obtained from leaves of J. virginiana. (a) -Pinene, (b) myrcene, (c) limonene, (d) linalool, (e) bornyl acetate, (f) safrole, (g) (Z)-iso-safrole, and (h) (E)-iso-safrole.
Safrole was reported as the major active constituent of essential oil of leaves obtained from Texas accessions (Von Rudloff, 1975), whereas that of Ontario had a low concentration of safrole (1.5%). The essential oil obtained in the present study (Fig. 3 and Table 3); shows a high concentration of safrole, i.e. 19.012 ± 3.8086%, closely followed by limonene (18.195 ± 0.6184%). Limonene was reported to be in high amounts in Ontario accessions of J. virginiana leaves with a concentration of 17.8%, whereas those collected from Texas produced only 0.9% of limonene. Another study on essential oil obtained from leaves of plant material collected from Oklahoma, reports the presence of 2.6% limonene (Dunford et al., 2007). In the present study the concentration of other components (-pinene, myrcene, linalool, (E)-iso-safrole, (Z)-iso-safrole and bornyl acetate) was low as compared to safrole and limonene. Previous reports found a variation in the content of these components from accessions collected from Texas, Ontario and Oklahoma, with 10.6% myrcene and 0.9% bornyl acetate being reported from Oklahoma accessions (Von Rudloff, 1975; Dunford et al., 2007), and 1.4% and 0.7% linalool from Texas and Ontario, respectively (Von Rudloff, 1975). The simultaneous extraction of essential oil and podophyllotoxin, without having to lose any podophyllotoxin during the steam distillation process is perhaps the most important finding of this study. J. virginiana is an evergreen and is abundantly available, to an extent that in some states it has been considered invasive (Gold et al., 2005). J. virginiana can be a consistent source of podophyllotoxin and Juniper leaf essential oil. Employing the present method of dual extraction of J. virginiana leaves offers an alternative source use for by-product, from which two commercially viable natural products can be obtained. In addition, we demonstrated that an approximately 6% pure product of podophyllotoxin could be obtained from the steam-distilled red cedar leaves. Although 6% purity is excellent for only a few steps, higher purity may be desirable for commercial
Table 3 Analysis of components of leaf essential oil of Juniperus virginiana. Sr. No.
Compound
1 2 3 4 5 6 7 8
Safrole Limonene -pinene Myrcene Linalool (E)-iso-safrole (Z)-iso-safrole Bornyl acetate
Concentration (%) 19.012 18.195 3.041 1.378 1.137 0.247 0.165 0.01
± ± ± ± ± ± ± ±
3.8086 0.6184 0.4264 0.2128 0.1505 0.1489 0.0566 0.00372
applications. Further research is needed to purify this product by column separations or precipitation experiments. 4. Conclusions This work demonstrated that both essential oil and podophyllotoxin can be extracted from the same biomass samples of J. virginiana leaves. It was found that the process of steam distillation of the essential oil did not degrade podophyllotoxin. Following the distillation of the essential oil, podophyllotoxin could be recovered from both the steam-distilled biomass and from the residual water. It was also found that J. virginiana heartwood (a traditional source for Juniper essential oil) does not contain podophyllotoxin. Acknowledgements Contribution of the Mississippi Agricultural and Forestry Exp. Sta. journal article No J-11568. This study was supported in part by USDA-NRI project “American mayapple and eastern red cedar as domestic sources for the anti-cancer compound podophyllotoxin”, and Mississippi State University/MAFES project “Bioprospecting for anti-cancer compound podophyllotoxin”. Authors thank the MAFES for continuous support. Authors also thank Amber Callahan for assistance with quantitative analysis. References Canel, C., Dayan, F.E., Ganzera, M., Khan, I.A., Rimando, A., Burandt, C., Moraes, M., 2001. High yield of podophyllotoxin from leaves of Podophyllum peltatum by in situ conversion of podophyllotoxin 4-O-b-d-glucopyranoside. Planta Med. 67, 97–99. Coleman, W.M., Lawrence, B.M., 1997. A comparison of selected analytical approaches to the analysis of an essential oil. Flavour Frag. J. 12, 1–8. Dunford, N.T., Hiziroglu, S., Holcomb, R., 2007. Effect of age on the distribution of oil in eastern red cedar tree segments. Bioresour. Technol. 98 (14), 2636– 2640. Eller, F.J., King, J.W., 2000. Supercritical carbon dioxide extraction of cedarwood oil: a study of extraction parameters and oil characteristics. Phytochem. Anal. 11, 226–231. Furnis, B.S., Hannaford, A.J., Smith, P.W.G., Tatchell, A.R., 1989. Vogel’s Textbook of Practical Chemistry, 5th ed. Longman Scientific & Technical, New York, NY, pp. 171–175. Gold, M.A., Godsey, L.D., Cernusca, M.M., 2005. Comparative market analysis of eastern red cedar. Forest Prod. J. 55, 58–65. Hartwell, J.L., Johnson, J.M., Fitzgerald, D.B., Belkin, M., 1953. Podophyllotoxin from Juniperus species; Savinin. J. Am. Chem. Soc. 75 (1), 235–236. Meijer, W., 1974. Podophyllum peltatum – mayapple: a potential new cash crop plant of eastern North America. Econ. Bot. 28 (1), 68–72. Payne, K.W., Wittwear, R., Anderson, S., Eisenbraun, E.J., 1999. Use of a modified abderhalden apparatus for comparing laboratory and industrial methods for
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isolating eastern red cedar essential oil. Forest Prod. J. 49, 90–92. Semen, E., Hiziroglu, S., 2005. Production, yield and derivatives of volatile oils from eastern redcedar (Juniperus virginiana L.). Am. J. Environ. Sci. 1, 133– 138. Von Rudloff, E., 1975. Volatile leaf oil analysis in chemosynthetic studies of North American conifer. Biochem. Syst. Ecol. 2, 131–167.
Zheljazkov, V.D., Cantrell, C.L., Tekwani, B., Khan, S., 2008. Content, composition, and bioactivity of the essential oil of three basil genotypes as a function of harvesting. J. Agric. Food Chem. 56 (2), 380–385. Zheljazkov, V.D., Avula, B., Jones, A.M., Maddox, V., Rowe, D.E., 2009. Lignan and nutrient concentrations in American Mayapple (Podophyllum peltatum L.) in the Eastern United States. HortScience 44 (2), 349–353.