Supercritical Fluid Extraction of Bioactive Components from St. John's

Chapter 9

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Supercritical Fluid Extraction of Bioactive Components from St. John's Wort (Hypericum perforatum L.) and Ginkgo biloba 1

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M a r i Mannila , Qingyong L a n g , Chien M. W a i , Yanyan C u i , and Catharina Y. W . Ang 2,*

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Department of Chemistry, University of Idaho, Moscow, ID 83844 Division of Chemistry, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, HFT-230, Jefferson, AR 72079

Supercritical fluid extraction (SFE) offers an attractive alternative to solvent-based methods for extraction and manufacturing of herbal products. In addition to being a "green" solvent, supercritical CO can also be selective for extraction and separation of active ingredients from certain herbs. The main active constituents in St. John's wort are hyperforin and adhyperforin, which can be effectively extracted with neat CO under mild conditions (30 °C and 80 atm). Furthermore, extraction of hyperforin and adhyperforin from St. John's wort with neat CO is selective, resulting in a fairly enriched and stable extract of these compounds. A successful supercritical CO extraction of terpene trilactones (bilobalide and ginkgolides A, Β and C) from Ginkgo biloba leaves can be achieved at an elevated temperature (100 °C) and high pressure (350 atm; 0.72 g/ml density) with a small amount of ethanol/acetic acid (9:1) as a modifier. SFE of bilobalide and ginkgolides under these conditions resulted in an approximately 10% higher yield compared with traditional solvent based extractions. 2

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© 2003 American Chemical Society

Gopalan et al.; Supercritical Carbon Dioxide ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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1. Introduction The use of herbal medicines and food supplements has gained increased popularity in recent years. The total vitamin, mineral, and herbal supplement market in 1999 was $14.1 billion in the U S A (1). According to a report i n 2000, the top purchased herbal products i n the U S A were gingko, garlic and glucosamine, followed by echinacea and St. John's wort (1). Bioactivity studies of these herbal extracts showed beneficial health effects. The extract of Ginkgo biloba was found to improve memory, having antioxidant activity and improving blood circulatory disorders (2). St. John's wort (Hypericum perforatum L.) is used as an antidepressant, antioxidant, and as a remedy for several psychological and neuralgic disorders (3-5). Although herbal products are widely used today, there is a lack of complete knowledge of the active constituents i n many herb extracts, their biochemical effects and possible interactions with other medicines. Commercially available herbal products usually have complex compositions and show significant variations in concentrations of active ingredients (6,7). It is known that medicinally active compounds often have positive health effects at a low dose but may be toxic at a high dose. Thus, large variations i n quality of herbal products could create health problems for the consumers. Therefore, developing a good knowledge of active compounds and their methods of analysis are crucial for safe use and quality control of herbal remedies. For analytical purposes, various organic solvents including methanol can be used for initial extraction of the herbs. Herb extracts intended for human consumption are generally prepared using organic solvents, such as alcohol or acetone. Extraction and processing of herbal products with a nontoxic and "green" solvent is highly desirable for consumer protection. Conventional solvent extraction methods have several inherent drawbacks including lack of selectivity and generation of liquid wastes. Supercritical fluid extraction (SFE) has gained acceptance i n recent years as an alternative to conventional solvent extraction for separation of organic compounds in many analytical and industrial processes. Carbon dioxide is widely used in SFE because of its moderate critical constants (T = 31 °C and P = 73 atm), nontoxic nature, and ready availability i n a relatively pure form. Thus, developing supercritical C 0 extraction methods for production of herbal products appear attractive with respect to economy, consumer protection, and environmental point of view. SFE has been applied to herb and natural product studies for years (8-10). This chapter describes procedures of optimization of S F E parameters for the separation of active constituents from two popular herbs: St John's wort and Ginkgo biloba. The active compounds in St. John's wort are believed to be c

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132 hypericins (77-73), hyperforins (14-16), and some flavonoids (77). Hyperforin, a major constituent i n S t John's wort, is a lipophilic phloroglucinol (Figure IA) . Adhyperforin, a homologue of hyperforin, is believed to have similar medicinal activities to hyperforin. These compounds are usually extracted from dried stem, leaves and flowers of S t John's wort using organic solvents such as methanol (18-19), acetone-ethanol (20), or hexane (20, 21). Earlier reports have shown that hyperforin and adhyperforin could be specifically extracted by supercritical C 0 (22). Larger-scale extraction methods with stabilizers or improved processing procedures were designed for production of S t John's wort extracts with high hyperforin content (23). However, no detailed information was available regarding the S F E conditions. Detection of hyperforin and adhyperforin has been performed most often by reverse phase H P L C combined with a diode array or ultraviolet detector. If the goal is to detect all the known active ingredients i n S t John's wort, a long analytical time (up to 90 min) with a gradient elution system is required (18, 20). If only hyperforin and adhyperforin are to be detected, a rapid H P L C procedure (about 15 min) with an isocratic solvent system can be used for quantitative analysis (19, 24).


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The main active ingredients i n Ginkgo biloba are believed to be bilobalide and ginkgolides, which are terpene trilactones having t-butyl groups (Figure IB) . Initially these compounds were isolated from the roots of Ginkgo biloba tree (25), but today the leaves of Ginkgo biloba are the main source of ginkgolides. Commonly used extraction solvents for separation of ginkgolides are water (26), acetone (27), and mixtures of water and ethanol (28). Several analytical methods including H P L C (29, 30), G C / M S (26), C E (57) (capillary elctrophoresis), and S F C (supercritical fluid chromatography) (28) have been developed for detection of ginkgolides (32). Among these methods, SFC with E L S D (evaporation light scattering detector) as an alternative analytical method showed some unique advantages, such as speed and decreased consumption of organic solvents. Furthermore, this method was less demanding in terms of sample preparation. However, the disadvantages are also obvious. For instance, higher requirements i n experience, instrument maintenance and relative standard deviation (RSD) values are greater than those of GC-FID (28). In addition to this, S F C with E L S D detector is not as easily available in many analytical laboratories as H P L C or G C . Most recently, Lang et al. reported a simple detection method based on gas chromatography (GC) connected to a flame ionization detector (FID) (8). In this procedure bilobalide and ginkgolides were first separated from the extract by liquid-liquid extraction followed by derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA, 99%) and trimethylsilyl chloride (TMCS, 1%), and finally analyzed by GC-FID.


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Hyperforin R=CH Adhyperforin R=CH CH 3


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3

B. cm

Bilobalide

Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J

RI R2 R3 R4 H O H H OH H OH OH OH OH OH OH O H OH O H H OH

Figure 1. Structures of (A) hyperforin and adhyperforin, and (B) bilobalide and ginkgolides.

2. SFE of Hyperforin in St John's Wort

2.1 Modifier Recent reports from our research groups have demonstrated that neat supercritical C 0 is selective for the extraction of hyperforin and adhyperforin in St. John's wort (24, 33) (Figure 2). Modifiers do not increase the S F E recoveries of these phloroglucinols, but tend to cause partial extraction of polar compounds in St. John's wort. Thus, extraction with neat C 0 is the best choice for selective removal of hyperforin and adhyperforin from St. John's wort. 2

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Figure 2. HPLC chromatograms of extract ofSt. John's wort by (A) SFE with neat C0 (40 Î7, 100 atm), and (B) ultrasonic extraction with methanol Peak 1: chlorogenic acid; 2; rutin; 3: quercitrin; 4:quercetin; 5: pseudohypericin; 6: hypericin; 7: hyperforin; 8: adhyperforin. (Reproduced with permission from reference 32. Copyright 2002 The Royal Society of Chemistry.) 2

2.2 Effects of temperature and pressure (density) Density is often an important parameter for achieving high extraction efficiency in the optimization of SFE conditions. Density of a supercritical fluid can be calculated from temperature and pressure of the fluid phase using computer models available in the literature. According to our experiments, the recoveries of hyperforin and adhyperforin from St. John's wort are good i f the density of the fluid phase is kept at 0.65 g/mL or higher i n the temperature range 30 - 90 °C and pressure range 80 - 300 atm. The density of a supercritical fluid decreases with increasing temperature at a given pressure. For example, at 100 atm pressure the density decreases from 0.85 g/mL to 0.41 g/mL when temperature rises from room temperature (22 °C) to 50 °C. This density decrease at elevated temperatures explains the decrease i n extraction efficiency of hyperforin and adhyperforin with increasing temperature at 100 atm as shown i n Figure 3 A . O n the other hand, at 300 atm the density of the fluid phase remains high (0.71 g/mL) even at 90 °C. Consequently, the extraction efficiency of hyperforin stays nearly constant at 300 atm i n the temperatures 30 - 90°C. A t 100 atm, a high extraction efficiency can be achieved at 40 °C due to a higher density under this condition (Figure 3B). Thus, very mild SFE conditions can successfully extract hyperforin and adhyperforin from St. Johns's wort.



t2J

Hfc

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60 70 Ttmp«ratur« (C)

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0.S

0.6 0.7 Density (gfm!)

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Rxed temperature (40 C)

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Figure 3. Effects of (A) temperature, pressure and β) density on the SFE efficiency of hyperforin in St John's wort with neat C0 (Reproduced with permission from reference 33. Copyright 2002 The Royal Society of Chemistry.)

\4 •

Extraction of Hyperforin


300 atm

200 atm


136 Temperature increase (at a fixai density of 0.65 g/mL) does not improve the extraction of hyperforin and adhyperforin i n St. John's wort. Maximum extraction was achieved at very mild conditions such as 30°C and 80 atm, even though these conditions are slightly below the critical point of the CO2. Because the change from liquid to supercritical fluid is known to extend over a small temperature range (several degrees), the property of liquid CO2 near the critical point is probably close to that of the supercritical state. Using mild conditions for extraction of natural products have several advantages including technical convenience, reduced co-extraction of undesirable products, and minimizing the risk of thermal decomposition of the desired compounds.

2.3 Extraction Time A relatively short extraction time (total of 20 min) is sufficient to complete the SFE of hyperforin and adhyperforin under optimized temperature and pressure conditions (33). Depressurization of the sample matrix during the extraction process could improve the over-all extraction efficiency. Most probably depressurization alters or breaks down the plant cells so that more solutes become extractable by the fluid. In extraction of 300 mg St. John's wort at 40°C and 100 atm, a 5 min static extraction followed by two 7 min (total 20 ml liquid C 0 ) dynamic extractions (with depressurization between dynamic steps) was sufficient to complete the extraction. Without depressurization (one 14 min dynamic extraction), about 90% of the maximum extraction could be achieved. 2

2.4 S F E compared to solvent extraction methods The efficiency of removing hyperforin and adhyperforin from St John's wort by S F - C 0 was studied by interlaboratoiy comparison between our two research groups. The SFE results were also compared to the ultrasound-aided extraction in methanol. The S F E results of hyperforin obtained by the two groups were equal (about 5 mg/g dry wieght), although different S F E conditions were used: 1) 50 °C and 375 atm (density > 0.9 g/ml), and 2) 30 C and 80 atm (0.65 g/ml). This is reasonable, since limiting factor for efficient extraction was found to be a fluid density of > 0.65 g/ml, that was reached with both S F E methods. Also the reproducibility of the S F E results was comparatively good; both research groups obtained relative standard deviations of 10-13% for hyperforin and adhyperforin. 2

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Compared to the methanol extraction, the SFE results were equal with one group, whereas another laboratory obtained higher efficiencies with methanol extraction. The SFE results were 70% of the higher methanol extraction result. Both groups used the same methanol extraction method (90 min i n ultrasound bath, after which the sample was centrifuged). It is possible that one ultrasound Gopalan et al.; Supercritical Carbon Dioxide ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

137 bath (PC620, Branson Cleaning Equipment Co., Shelton, CT) was more effective than the other (FS30, Fisher Scientific, Suwanee, G A ) and thus hyperforin and adhyperforin were separated more efficiently by the former. Due to the higher efficiency of methanol extraction, S F E can not be used for the quantitative analysis of hyperforin and adhyperforin i n St. John's wort. The selectivity of supercritical C0 extraction of these phloroglucinols is much better than most of the organic solvents (33). The peak area of hyperforin and adhyperforin in the crude extract obtained from the SFE process was 85% (69% hyperforin and 16% adhyperforin) of the all peaks detectable by H P L C / U V . Only hexane shows selectivity similar to that of C 0 , but the use of SFE is beneficial due to its non-toxic nature. Furthermore, the stability of hyperforin was found to be poor in non-polar organic solvents such as hexane (34). Concentrating St. John's wort extract i n hexane was shown to be a main source for peroxide formation that led to the oxidation and decomposition of hyperforin (21). SFE extracted compounds can be collected i n a small amount of suitable solvent (such as ethanol or methanol) and thus there is no need for sample concentration and evaporation of solvents. This can be a very crucial advantage in isolation of phyto-nutraceuticals, because these compounds are often veiy air- and light-sensitive and thus can easily decompose during sample treatment.


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Hyperforin and adhyperforin have been reported to undergo decomposition almost completely (over 90%) in methanol/acetonitrile (1:9) solution when they were exposed to light and air for 2 hr after mixed solid-phase purification (19). Different research groups have achieved different results of the stability of hyperforin i n methanol solution. According to one report, 96% of hyperforin was decomposed i n 5 min when methanol extract of St. John's wort was exposed to direct daylight (35). On the other hand, only 40% decomposition was observed after 30 days when purified hyperforin was stored i n daylight i n methanol solution (34). Decomposition of hyperforin also depends on p H : storage in methanol with strongly basic conditions (pH 12) caused complete decomposition of hyperforin during 30 (toys i n daylight (34). Thus, decomposition of hyperforin can be minimized by 1) protecting extract from light and air, 2) using polar storage solvents, 3) using nonbasic p H , and 4) using minimal sample treatment after extraction. When the crude SFE extract was dissolved in methanol, no decomposition of hyperforin and adhyperforin was observed even after 80 hr of exposure to direct daylight and air (33). During SFE, hyperforin can be trapped i n an empty vial and dissolved i n a small amount of polar solvent, so there is no need for evaporation of solvent after extraction. Furthermore, in the crude SFE extract, some impurities may protect hyperforin from oxidation, and probably the SFE procedure may inhibit peroxide formation. Because the isolation of hyperforin and adhyperforin from SFE extract can be achieved with minimum sample treatment and, consequently, with reduced exposure to air, the decomposition of the hyperforin can be minimized.


138 2.5 Additional studies of hyperforin and adhyperforin i n S F E extract Because SFE of St. John's wort with neat C 0 gives a comparatively high concentration and stable mixture of hyperforin and adhyperforin, the crude SFE extract can be used directly for certain studies to improve the knowledge of these compounds. For example, crude SFE extract has been used successfully to study the tautomerism of the hyperforin (33) and i n animal studies for investigation of bioactivities (36-38). Hyperforin is a colorless, oily, and easily decomposed compound i n its pure form, and thus the pure form is difficult to isolate. Orth et al. has reported a preparative-HPLC isolation procedure of hyperforin from solvent extract (21). The crude SFE extract of St. John's wort using our method may offer increased recovery i n the isolation of these compounds in pure form using preparative H P L C . The SFE crude extract which contained approximately 30 to 40% of hyperforin and adhyperforin on a dry weight basis can be purified to different degrees of purity for different purposes. For example, i n one of our studies the crude extract i n methanol was partitioned with hexane saturated with methanol to remove co-extracted nonpolar substances. The hexane layer was discarded and the methanol layer was evaporated under vacuum and the residue was dissolved i n hexane for subsequent clean-up through a silica gel column. Impurities were washed out by hexane and hyperforins were eluted by hexanerethyl acetate (9:1). The eluted solution was mixed with 10-20 m L methanol and evaporated to near dryness under vacuum. The residue contained about 90-93% hyperforins. It can be dissolved in ethanol for further use i n pharmacokinetic and in vivo drug-herb interaction studies. For the metabolic study i n our laboratoiy, the residue was further purified by analytical H P L C and resulted i n isolated hyperforin and adhyperforin fractions at 98% purity. The purified hyperforin was used i n invitro rat liver microsome systems designed to elucidate biotransformation mechanism of hyperforin and the major metabolic pathway. More than 10 metabolites were found by H P L C / P D A method and 4 major metabolites were isolated and identified by L C / M S and N M R (39).


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3. SFE of Ginkgolides from Ginkgo biloba

3.1Modifier Supercritical C 0 is a poor extraction media for intermediately polar to polar compounds and thus a polar organic modifier is needed to improve the extraction of bilobalide and gingkolides from Gingko biloba. Without a modifier, no ginkgolides were detected from the S F E extract. Methanol is the 2



139 most often used modifier in SFE, but it is not suitable i n extraction of food supplements due to its toxicity. Thus, a less toxic mixture of solvents, ethanol and acetic acid (9:1), was selected as a modifier to enhance the extraction of bilobalide and ginkgolides in supercritical CO2. According to our experiments, a small amount of modifier (0.5 ml per gram of leaves) could significantly improve the SFE efficiency of the terpene trilactones. The highest S F E efficiency was achieved with two times of SFE with total of 5 m L modifer (3 + 2 mL) per gram of ginkgo leaf powder at 100 ° C and 3 5 0 atm. In fact, at the beginning of the SFE process, there were two phases (extra modifier liquid and supercritical fluid) existing inside the SFE vessel. This is necessary i n order to extract the more polar ginkgolide C as well as to prevent tubing clogging. Extraction of ginkgolides using boiling ethanol requires more than 4 0 m L of the solvent per gram of the leaf powder to achieve a similar efficiency.

3.2 Analysis of ginkgolides After SFE, the extract was diluted with ethanol i n a 2 5 - m L volumetric flask, and 5 mL of the solution was used for analysis. The solvent was first removed by evaporation i n a warm water bath, and then the ginkgolides were dissolved in 5 mL of 5 % N a H P 0 aqueous solution. In order to save time, the aqueous solution may be centrifugea to settle down the insoluble residue. Precisely 1 m L of the clear supernatant was transferred into a small sample vial, and 3 mL of ethyl acetate/tetrahydrofuran ( 7 0 : 3 0 v/v) and 2 5 μg of squalane (in hexane) as an internal standard were added into the vial for liquidliquid extraction separation. The vial was shaken for 1 min either using a mechanical shaker or manually. After phase separation, about 1 - 2 m L of the organic solution was transferred to a dry and clean sample vial for derivatization. The solvent was blown down to dryness with a gentle stream of nitrogen gas in a hood and 3 0 0 j i L of D M F and 3 0 0 H L of B S T F A / T M S C ( 9 9 : 1 ) were added to the vial. The capped vial was placed i n a G C oven at 1 2 0 °C for 4 5 min for derivatization. The standards of ginkgolides were treated i n the same way. After cooling down to room temperature, 1 \£L of each sample was injected into a G C for quantification. 2

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For G C analysis, helium was used as the carrier gas and the head pressure was set at 1 8 psi. The injector was set at 2 8 0 °C and the detector at 3 0 0 °C. The oven was programmed to have an initial temperature of 2 0 0 °C for 1 min, and was then ramped to 2 8 0 °C at a rate of 1 0 °C/min and held at the final temperature for 5 min. The details of the analytical procedure are given in the literature (8).


140 3.3 Effect of temperature and pressure (density) Increasing the extraction temperature has been reported to improve the SFE efficiency for many organic compounds (40) including certain natural products from plant matrices. According to our studies, increase i n extraction temperature (at 350 atm) improved the extraction of the terpene trilactones (Figure 4). Higher températures can lower the viscosity and increase the difixisivity of the fluid Thus, the fluid can penetrate into the leaf sample and reach the extractable compounds located deeper inside the matrix. Furthermore, a higher temperature tends to increase the vapor pressure of solutes and thus facilitates the transfer of the solutes from the matrix to the fluid phase. The maximum extraction efficiency for bilobalide and ginkgolides was achieved at a temperature around 100 °C. A t temperatures greater than 120 °C, the extraction efficiency was found to decrease. This might be related to the lower fluid density or due to decomposition of the solutes. Thus, 100 °C appeared to be a suitable extraction temperature for ginkgolides from ginkgo leaves.


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o-i 40

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80

100

120

Temperature (°C)

Figure 4. Temperature effects on the SFE results of terpene trilactones.

Raising pressure at a fixed temperature improves the extraction efficiency due to an increased fluid density. In the extraction of bilobalide and ginkgolides, the maximum extraction efficiency was achieved when the density was at least 0.72 g/mL. Because the extraction of bilobalide and ginkgolides is improved at elevated temperature, high pressures are required to obtain the minimum needed density. A t the optimal temperature (100 °C), a density of 0.72 g/mL was obtained at 350 atm pressure. With the optimized conditions (ethanol/acetic acid as a modifier, 100 °C and 350 atm), quantitative extraction of terpene trilactones from l g sample of Ginkgo biloba was achieved with 20 min static and 20 min dynamic extraction times (two times of SFE). Under


141 these conditions, the total amount of B - B , G - A , G-B and G - C extracted was about 4.7 mg per gram of the dry ginkgo leaves used in this study.


3.4 S F E compared to the solvent extraction methods The SFE technique was compared to the boiling liquid methods performed with the following solvents: 1) acetone, 2) ethanol, 3) methanol, 4) water, and 5) water/methanol (70:30 v/v). The SFE method showed the highest extraction efficiency and reproducibility (RSD

Supercritical Fluid Extraction of Bioactive Components from St. John's

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