Extraction of hyperforin and

Recent teports from our research groups have demonstrated that neat supercritical CO2 is selective for the extraction of hyperforin and adhyperforin in St. John s wort (24, 33) (Figure 2). Modifiers do not increase toe SFE recoveries of these phloroglucinols, but tend to cause partial extraction of polar compounds in St. John s wort. Thus, extraction with neat CO2 is toe best choice for selective removal of hyperforin and adhyperforin from St. John s wort. 

Extracts of hypericum may vary considerably in terms of the quantity and ratio of their constituents based on the extraction process used. Maximum extraction of hypericin and pseudohypericin is obtained with an 80% methanol solvent at 80°C (Wagner and Bladt 1994). Hyperforin is a lipophilic constituent of hypericum that is present in the oil extract (Chatterjee et al. 1998a). It is not very stable, but its presence is sustained by hot maceration of the flowers and storage in the absence of air (Maisenbacher and Kovar 1992). 

The pharmacokinetics of hyperforin have been studied in rats and humans (Biber et ai. 1998). In rats, after a 300 mg/kg orai dose of hypericum extract (WS 5572, containing 5% hyperforin), maximum piasma ieveis of 370 ng/mi (690 nM) are achieved at 3 hours. The haif-iife of hyperforin is 6 hours. Humans given a 300 mg tabiet of hypericum (containing 14.8 mg hyperforin) showed maximum piasma ieveis of 150 ng/mi (280 nM) at 3.5 hours. The haif-iife is 9 hours, and mean residence time is 12 hours. Pharmacokinetics of hyperforin are iinear up to 600 mg, and no accumuiation occurs after repeated doses. By comparison, effective and safe piasma ieveis of paroxetine and fluoxetine vary between 40 and 200 ng/mi (Preskorn 1997). The effective piasma concentration of hyperforin predicted from computer-fit data is approximateiy 97 ng/mi (180 nM), which couid be easiiy monitored (Biber et ai. 1998). There is a iinear correiation between orai dose of hyperforin and piasma ieveis, and steady-state concentrations of 100 ng/mi (180 nM) couid be achieved with three-times-daiiy dosing. 

Emit of detection was 4 ng mL and the limit of quantitation was 10 ng mL Bauer et al. described HPLC combined with UV detection for the determination of hyperforin and HPLC combined with fluori metric detection for the determination of hypericin and psudohypericin in human plasma. They used liquid-liquid extraction. The limit of quantitation was 10 ng mL for hyperforin and 0.25 ng mL for both hypericin and pseudohypericin. 

A relatively short extraction time (total of 20 min) is sufBcient to complete the SFE of hyperforin and adhyperforin under optimized temperature and pressure conditions (3S). 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 Ity two 7 min (total 20 ml liquid CO2) dynamic extractions (with depressurization between (tynamic steps) was sufficient to complete the extraction. Without dqrressurization (one 14 min dynamic extraction), about 90% of the maximum extraction could be achieved. 

ESI-MS in negative mode of Hypericum perforatum extracts provides typical fingerprints showing three different classes of compounds, i.e., flavonoids, hypericins, and hyperforins (Fig. 2). The abundant ions at m z 535 and 549 are due to [M-H] ions of hyperforin and adhyperforin, respectively. Deprotonated molecules ([M-H] ) of hypericin m z 503), pseudohypericin (miz 519), and... 

Amino acid neurotransmitter Constituents of hypericum also appear to have effects on amino acid neurotransmission, particularly GABA. Hypericin and a crude extract bind to GABAA and GABAB receptors (Cott 1997). Hyperforin also inhibits synaptosomal GABA reuptake in the low micromolar range (IC50 values of 0.05-0.10 ug/ml). Activity at GABAA benzodiazepine receptors was noted in extracts of four hy-... 

Electrophysiological effects Extracts of hypericum were examined for their electrophysiological effects in animals. The onset of effects occurred 3-4 hours after administration. Frequencies affected first were in the alpha range and were maximal in the frontal cortex (Dimpfel and Hofmann 1995). Another study examined the EEG effects for two hypericum extracts in rats one extract high in hyperforin and lacking naphthodi-anthrones (C02), and another extract (LI 160) low in hyperforin. Both extracts showed similar early alpha effects, but only LI 160 had a late effect of increased delta frequencies. The alpha effects are comparable to... 

Biber A, Fischer FI, Romer A, Chatterjee SS. (1998). Oral bioavailability of hyperforin from hypericum extracts in rats and human volunteers. Pharmacopsychiatry. 31(suppl 1) 36-43. 

Dimpfel W, Schober F, Mannel M. (1998). Effects of a methanolic extract and a hyperforin-enriched C02 extract of St. John s Wort (Hypericum perforatum) on intracerebral field potentials in the freely moving rat (Tele-Stereo-EEG). Pharmacopsychiatry. 31(suppl 1) 30-5. 

It is not possible to discuss pharmacokinetics when the active compound or compounds of St. John s wort are not known. The half-life of hypericin and hyperforin have been estimated at between 6 and 9 hours, with peak plasma concentrations at about 2-3 hours after administration. Some of the ingredients of Hypericum extracts are metabolized in the liver. 

As with drugs and purified biomarkers, thermal- and photostability of botanical products are the factors that must be considered. Commercial dried extract and capsules of SJW were evaluated under harmonized test conditions (25). Photostability testing showed all the constituents to be photosensitive in the tested conditions. However, different opacity agents and pigments influenced the stability of the constituents. Amber containers had little effect on the photostability of the investigated constituents. Long-term thermal stability testing showed a shelf life of less than four months for hyperforins and hypericins, even when ascorbic and citric acids were added to the formulation. 

Figure 4 SJW extracts and hyperforin induce CYP3A4 expression in human hepatocytes. Northern blot analysis was performed with total RNA (10 pg) prepared from primary cultures of human hepatocytes treated for 30 hours with extracts prepared from three different commercial preparations of SJW [extract 1, Nature s Way (9 pg/mL) extract 2, Nature s Plus (75 pg/mL) extract 3, Solaray (7 pg/mL)], 1 pM hyperforin, or vehicle alone (0.1% ethanol). The blot was probed sequentially with P-labeled fragments of CYP3A4 and fi-actin. Abbreviations SJW, St. John s wort CYP, cytochrome P450. Source From Ref 42.

Single- and multiple-dose pharmacokinetic studies with extracts of SJW were performed in rats and humans, which focused on the determination of plasma levels of the naphthodianthrones hypericin and pseudohypericin and the phloroglucinol derivative hyperforin. Results from pharmacokinetic... 

Human clinical studies Plasma levels of hyperforin were followed for 24 hours in two studies with healthy volunteers after administration of film-coated tablets containing 300 mg SJW extract representing 14.8 mg hyperforin (Table 2) (72). In the first crossover study, six male volunteers received 300, 600, or 1200 mg of a SJW extract preparation (WS 5572, Dr. Willmar Schwabe Arzneimittel, Karlsruhe, Germany) after a 10-hour fasting time. Maximum plasma levels of 150 ng/mL (approximately 280 nM) were reached after 3.5 hours after intake of 300 mg SJW extract. Half-life and MRT were 9 and 12 hours, respectively. Hyperforin pharmacokinetics were linear up to 600 mg of the extract. Increasing the doses to 900 or 1200 mg resulted in lower Cmax and AUC values than those expected from linear extrapolation of data from lower doses. In a repeated dose study with seven healthy volunteers, no accumulation of hyperforin in plasma was observed after intake of 900mg/day SJW extract for seven days. The estimated steady-state plasma concentrations of hyperforin after intake of 3 x 300mg/day was approximately 100 ng/mL (approximately 180 nM) (Table 2) (72). 

Animal studies Pharmacokinetics of hyperforin after administration of an ethanolic SJW extract (WS 5572, Dr. Willmar Schwabe, Karlsruhe, Germany) to rats were investigated by Biber et al. (72). Maximum plasma levels of approximately 370ng/mL (approximately 690 nM) were reached after three hours. Estimated half-life and clearance values were six hours and 70mL/min/kg, respectively. 

To ensure lot-to-lot consistency, standardization of extracts often relies on constituents as biomarkers for plant identity and potency. SJW Hypericum perforatum), a perennial shrub traditionally used as a mood enhancer and mild antidepressant, has been tested in dozens of clinical trials, with mixed results for efficacy. Some of its purported bioactive constituents include naphthodianthrones, including hypericin flavonoids phloroglucinols, including hyperforin and essential oils. For many years, hypericin was presumed to be the active component. As a result most extracts were standardized based on hypericin concentration. Recent data, however, support other components such as hyperforin and the flavanoids, that may also contribute to the therapeutic efficacy of the SJW extracts (33-35). Because these secondary components were previously unaccounted for in the standardization of the former clinical test articles, and because these constituents are chemically unrelated to and their content within the plant varies independently of hypericin, it has been argued that the potency of these constituents in any particular batch was unlikely to be similar to that of other batches. This variability between batches could explain the observed differences in the clinical trial results (36). 

Quantification of Hyperforin in Mice Brain by Liquid-Liquid Extraction and High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry, Using External Calibration (No Internal Standard)... 

Another common plant that appears to have some activity against MRSA is Hypericum perforatum L., commonly known as St. John s wort. In traditional folk medicine, oily extracts of St. John s wort are used for topical treatment of wounds, bums and myalgia [39]. The lipophilic phloroglucin-derivative hyperforin. Fig. 3, has antibacterial effects and has been shown to inhibit the growth of MRSA at a concentration of 1 (ig/ml [39]. Thus, investigating known plants based on their historical medical (ethnomedical) use is a potentially useful means of discovering new alternative antimicrobial agents. 

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