Extraction of rosavin from Rhodiola rosea root using supercritical carbon dioxide with water

J. of Supercritical Fluids 50 (2009) 29–32

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Extraction of rosavin from Rhodiola rosea root using supercritical carbon dioxide with water Pamela Iheozor-Ejiofor, Estera Szwajcer Dey ∗ Lund University, Pure and Applied Biochemistry, Box 124, 221 00 Lund, Sweden

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Article history: Received 24 September 2008 Received in revised form 18 April 2009 Accepted 19 April 2009 Keywords: Rhodiola rosea Rosavin Bioactive substances Supercritical carbon dioxide (scCO2 ) with water Solvents Extraction HPLC

a b s t r a c t In this study, a new extraction method for the isolation of rosavin from dried crushed roots of Rhodiola rosea is being developed using supercritical CO2 and water. Rosavin extracts quantitatively and qualitatively were compared to commonly used solvents such as methanol, ethanol and ethyl acetate. By HPLC analysis rosavin was found to be the dominant compound in extracts obtained by both extraction methods. Quantitative differences were observed between the two extraction methods. Among the solvents, methanol yielded 3.3% while ethanol only 1.2% of rosavin. Supercritical CO2 and water at extraction temperature 80 ◦ C and 5 h yielded 4.5% of rosavin. © 2009 Elsevier B.V. All rights reserved.

1. Introduction A variety of high value added products present in plant material can be successfully processed in supercritical fluids. Roots are a typical matrix from which bioactive compounds are extracted. Some of the natural resources with very high value bioactive substances are the roots and rhizomes of Rhodiola rosea, an herbaceous plant of the Crassulaceae family. They were traditionally used in Asia and Europe, especially in the Artic region for medicinal purposes [1,2], but with limited research. Several studies have been carried out on the plant, of its adaptogenic properties, and bioactivity components. These properties include inhibition of acetyl cholinesterase [3], anti-stress and anti-cardiac damage effect [4], in cancer therapy [5], as an antidepressant [6] or antioxidant [7], reduction of mental fatigue [8] chemo-preventive and/or therapeutic agent in the treatment of type II diabetes and hypertension [9]. Hence this plant is a favorite product for the development of wholesome foods, nutraceuticals, pharmaceutical and cosmetics. The effects mentioned are attributed to the phenylpropanoids, organic acids and flavonoids of the plant such as; rosavin (cinnamyl6(6 -0-␣-l-arabinopyranoside)-0-␤-d-glucopyranoside), salidroside(2-(4-hydroxyphenyl)ethyl-0-␤-d-glucopyranoside) and its aglycon tyrosol, rosarine(cinnamyl-␤ -0-␣-l-arabinofuranosyl)-0␤-d-glucopyranoside); rosin (cinnamyl-0-␤-d-glucopyranoside),

∗ Corresponding author. Tel.: +46 46 222 8258; fax: +46 46 222 4611. E-mail address: [email protected] (E.S. Dey). 0896-8446/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2009.04.011

gallic acid and rhodioflavanoside. Several clinical research works have made use of commercial standardized extract of 3–5% rosavin and 1% salidroside [10–12]. All the bioactive compounds are amphiphilic. The increasing demand of bioactive substances from rose-root leads to its destructive exploitation. The whole plant must be destroyed in each harvest after 5-years of growth. Consequently, an efficient and environmentally friendly isolation method is called for. Supercritical CO2 (scCO2 ) method is the most attractive and environmentally friendly method of choice. Overall, our and others results demonstrate comparable and often with high recoveries of bioactive compounds from the scCO2. The extracts generated by scCO2 contains lower quantities and number of interfering compounds [13–15]. This method is already accepted in industrial scale in food and pharmaceutical applications. Its qualities are: low cost, low toxicity, solvent-free product, low reactivity and non-flammability with attraction to hydrophobic compounds. Therefore, for the isolation of amphiphilic compounds, CO2 has to be combined with a polar co-solvent [16]. The most commonly used co-solvents are: ethanol and methanol. Water has been very successfully used for the isolation of lipopolysaccharides [17]. The study focuses on the extraction of rosavin using scCO2 by changing process factors such as extraction temperature, time, and co-solvent type and concentration. The results from scCO2 based method were compared to conventional solvent extraction. The yield of rosavin was monitored by HPLC and compared with a commercial standard.

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2. Material and methods 2.1. Chemicals The rosavin standard was from ChromaDex. HPLC grade acetonitrile (99.9%) and methanol (99.9%), LAB-SCAN Dublin, Ireland. Ethanol (99.5%) Kemetyl (Haninge, Sweden); analytical grade sulphuric acid, ethyl acetate (99.7%) Sigma–Aldrich (Steinheim, Germany); chloroform and ortho-phosphoric acid (analytical grade) provided by Merck (Darmstadt, Germany) and HPLC grade diethyl ether Fluka. Milli-pore water (18.2 M) Maxima Ultra pure water (Abino Lab.) 2.2. Samples Dried crushed root of R. rosea were provided by Gösta Lilius courtesy of SWEPHARM AB, Sweden. 2.3. Solvent extraction The crushed root was sieved to obtain a particle fraction of 0.5–1.0 mm in size. 2 g of dried root samples were extracted with 16 ml of solvent (ethanol, methanol or ethyl acetate) in a parafilm sealed beaker with magnetic stirring (50% of the max scale rpm) for 3 h. Samples were later carefully decanted and the spent roots rinsed with the same volume of fresh solvent. The extracts were pooled and filtered, centrifuged at 6000 rpm (4960 × g) for 10 min and the supernatant carefully decanted and collected. The organic  chner solvents were removed on rotary evaporator at 55 ◦ C (Bu rotarvapor R-200). The dry masses of crude extracts were weighed and reconstituted with ethanol. All extractions were done in triplicates. 2.4. ScCO2 extraction The rosavin from crushed root with particle size fraction of 0.5–1.0 mm (Section 2.3) was extracted by supercritical CO2, using the Thar SFE 100X2F system (Thar technology Inc., Pittsburgh, PA, U.S.A.). The schematic diagram of the original system is shown in Fig. 1. In the present studies only the extraction vessel 2 (V2), and both cyclone collectors (CS1 and CS2) were used. For each extraction, 5 g

of sample was placed and covered with a small piece of glass wool, to prevent loss/bumping of samples and subsequent blocking of the extraction pipes, before closing the extraction vessel. The carbon dioxide pressure was adjusted to 20 MPa. This fixed pressure was used in all trials. The temperatures were kept; at 70 and 80 ◦ C and the extraction time was 3 and 5 h. In all trials carbon dioxide flow rate was kept constant at 9 g/min, and co-solvents concentration as carbon dioxide percentage, at 10%. Co-solvents details are given in the text of the corresponding tables. All co-solvents used were degassed. The extracts collected from cyclone/separator were filtered, and centrifuged at 6000 rpm (4960 × g) for 10 min. The organic cosolvents were removed on rotary evaporator at 55 ◦ C (Bu  chner rotarvapor R-200), and the samples extracted with water as cosolvent were freeze dried (Ab Nino lab. Sweden). The extracts were reconstituted in ethanol. Each extraction was performed in triplicates and the samples were stored at 4 ◦ C before analysis. The standard deviation of n measurements was calculated by applying “Statistics of repeated measurement” [18]. 2.5. High performance liquid chromatography (HPLC) All samples were analyzed using Agilent HP 1050HPLC equipped with a UV detector and an auto sampler. A reversed phase Kromasil C18 (250 mm × 4.0 mm), 5 ␮m particle size column (Chromatech AB) was used and controlled by ChemStation software (Agilent Technologies). ChromaDex protocol (2005) was used with a slight modification due to differences in HPLC equipments and minor problems encountered during analysis. Prior to use, all mobile phase were vacuum degassed and samples were passed through 0.2 ␮m Acrodisc syringe filter from Pall (Ann Arbor, MI, U.S.A.). The UV wavelength was kept at 254 nm. Five microliters of extract (0.1 mg/ml) was injected in triplicates with an auto sampler and elution was conducted at ambient temperature. 0.2% (v/v) phosphoric acid/acetonitrile (96:4, v/v) was used as the mobile phase at a flow rate of 0.6 ml/min for 20 min. After 20 min, elution continued for 30 min with 0.2% (v/v) phosphoric acid/acetonitrile (70:30, v/v) at a constant flow rate. Rosavin standard in methanol (0.5, 1, 1.5, 2.0 and 2.5 ␮g) was used. To determine the peak identity, the extracted samples were also spiked with 12 ␮l of the standard rosavin sample.

Fig. 1. Schematic diagram of the supercritical fluid extractor Thar SFE 100X2F.

P. Iheozor-Ejiofor, E.S. Dey / J. of Supercritical Fluids 50 (2009) 29–32

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Fig. 2. HPLC chromatograms. Rosavin standard (A), extract from Table 3, row 5 (B), ethanol extract (C) and methanol extract from Table 1 (D). The HPLC conditions are described in Section 2.5.

Table 1 Gravimetric and HPLC data of samples extracted from Rhodiola rosea with organic solvents. The amount of crude extract and rosavin are expressed as mg in gram of dried root. The extraction conditions are in Section 2.3. Solvent Methanol Ethanol Ethyl acetate

Crude extract (mg/g dried root) 117.5 50 30

(n = 3)  (±)

Rosavin content (mg/g dried root)

(n = 3)  (±)

5 5 4

33.0 12.00 11.23

0.6 0.28 0.02

3. Results and discussion

3.2. Quantification

3.1. Identification

The crude extracts and rosavin yields are expressed as milligrams of crude extract and rosavin/g of dried weight roots, respectively. Table 1, Fig. 2C and D shows results from solvent extraction and in Tables 2 and 3, Fig. 2B from scCO2 co-solvents extraction. Methanol and ethanol are commonly used solvents for the extraction of rosavin from roots. For food based application ethanol is the favorable solvent in spite of the facts that the yields are 50% lower than with methanol (Table 1). For a better yield and solvent-free products, scCO2 was chosen as an alternative method. Rosavin possess amphiphilic properties, therefore polar co-solvent such as water, methanol and ethanol were tested (Table 2). Contrary

HPLC analysis was carried out for the identification and quantification. Rosavin standard was used for the identification of peaks in extracts made by organic solvents and scCO2 . The chromatogram of the standard (Fig. 2A) shows that rosavin is eluted at retention time of 37 min. Fig. 2B–D represents chromatograms of selected extracts that exhibit similar patterns with the predominant peak at the retention time of 37 min, belonging to rosavin. Additional peaks at retention times 14, 38 and 40 min are present in all the samples.

Table 2 Gravimetric and HPLC data of samples extracted from Rhodiola rosea with scCO2 using different co-solvents. The amount of crude extract and rosavin are expressed as mg in g dried root. ScCO2 flow 9 g/min; pressure, 20 MPa and 10% co-solvent, extraction time, 3 h; temperature, 70 ◦ C. Co-solvent

Crude extract (mg/g dried root)

(n = 3)  (±)

Rosavin content (mg/g dried root)

Water Methanol Hydro-ethanolic (ethanol/water 4:1) Hydro-ethanolic (ethanol/water 1:1)

210 30 20 230

4 2 2 4

33.17 0.42 0.13 27.27

(n = 3)  (±) 0.20 6e−3 4e−3 0.20

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Table 3 Effects of temperature and time on the yield of crude extracts and rosavin obtained with scCO2 using water as co-solvent. ScCO2 flow 9 g/min; pressure, 20 MPa and 10% co-solvent. Temperature (◦ C)

Time (h)

Crude extract (mg/g dried root)

(n = 3)  (±)

Rosavin content (mg/g dried root)

(n = 3)  (±)

70 70 80 80

3 5 3 5

210 198 184 276

4 7 3 3

33.17 42.9 42.45 45.51

0.20 0.17 0.20 0.13

to conventional extraction (Table 1) pure methanol as co-solvent, shows very low yield of crude extract and rosavin. Higher co-solvent stock concentrations have been reported to carry negligible effect [19]. Lin et al. [20] observed better extraction yields using diluted methanol (70%) against pure methanol and viewed a combined negative effect using pure methanol at temperature above 50 ◦ C. Ethanol diluted with water, in the same proportion (1:1) facilitates in the increase of crude extract and rosavin. The best yield of rosavin was obtained with pure water as co-solvent (Table 2). This seems to be due to the ability of water to interact with the polar ends of the sugar moieties of rosavin having its polarity. Moreover, water can; (i) increase the bulk density of fluid mixture [21] or (ii) soften and subsequently swell the sample matrix made up of lignin and cellulose, thereby altering the matrix–analyte diffusion process favoring scCO2 penetration [22] for better solubility enhancement. From our previous experiences in using scCO2 for the isolation of bioactive substances, we gained much higher levels of extracted compounds by scCO2 than by the established conventional methods [13,15,17]. As majority of bioactive substances are amphiphilic, CO2 has to be combined with a polar co-solvent [16]. Ethanol and methanol are the commonly used co-solvents. Water has seldom been used, however it was successfully used by us in the isolation of lipopolysaccharides [17]. Rosavin molecule (cinnamyl-6(6 0-␣-l-arabinopyranoside)-0-␤-d-glucopyranoside) constitutes a predominant sugar moiety, therefore the favor of using water as co-solvent seemed to be logical. From Table 2 (last row and second row) increase of the crude extract size was observed, which may originate from the endogenous water present in the root. Nevertheless, the rosavin yield is strongly improved with pure water as co-solvent. The extractability effect of temperature (70 and 80 ◦ C) and extraction time (3 and 5 h) were tested (Table 3) for the maximum yield. In extending the extraction time from 3 to 5 h (at 70o C) or increasing the extraction temperature (3 h) almost 25% more rosavin was extracted. The increase of extraction temperature (80 ◦ C, 3 h) accelerated the extraction of rosavin. Since no increase of rosavin was gained in the extended time (5 h), offering an indication of exhaustion. Different pressures in combination with co-solvents were not tested, since already under the used pressure (20 MPa) and water as co-solvent (Table 3) much higher amounts of rosavin was obtained than from the methanol extraction (Table 1). An accumulative sampling might optimize the extraction time and smaller fraction size of the root may increase the accessibility of the solvents, but at this stage we have no indicative reference to monitor the complete exhaustion of rosavin in the tested material. From this work we can conclude that at low cost with high yields food grade rosavin can be obtained. The developed method will be adopted for the estimation of rosavin in the selection, preservation and conservation of genetic resources of R. rosea [23]. For adoption of the current extraction method, effect of water content in the micropropagated and fresh root and other factors has to be considered. Acknowledgment This study was partially supported by Kemira Oyj, Finland.

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