Extraction pseudohypericin

Fig. 2.49. Profile of Hypericum perforatum extract with the H LC-MS attributions of the components detected. 1 = chlorogenic acid isomer 2 = 3-0- -coumaroylquinic acid 3 = chlorogenic acid 4 = rutin 5 = hyperoside 6 = isoquercitrin 7 = 3,3, , , 7-pentahydroxyflavanone 7-0-rhamnopyranoside 8 = quercitrin 9 = quercetin 10 = 13,118 tapigenin 11 = pSeudohypericin 12 = hypericin 13 = hyperforin analogue 14 = hyperform dialogue 15 = hyperforin 16 = adhyperforin. Reprinted with permission from M. Brolis eta. [ ].

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 hypericin and pseudohypericin piasma have been studied as weii (Brockmoiier et ai. 1997). Human subjects receiving piacebo, or 900, 1800, or 3600 mg of a standardized hypericum extract (LI 160), which contained 0, 2.81, 5.62, and 11.25 mg of totai hypericin and pseudohypericin, achieved maximum total plasma concentrations at 4 hours (0.028, 0.061, and 0.159 mg/L, respectively). The half-lives of absorption, distribution, and elimination were 0.6, 6.0, and 43.1 hours, respectively, using 750 pg of hypericin, and are slightly different for 1578 pg of pseudohypericin (1.3, 1.4, and 24.8 hours, respectively) (Kerb et ai. 1996). The systemic availability of the hypericum extract LI 160 is between 14 and 21%. Comparable results are found in another study using LI 160 (Staffeldt et ai. 1994). Long-term dosing of 3 x 300 mg per day showed that steady-state levels of hypericin are reached after 4 days. 

Staffeldt B, Kerb R, Brockmoller J, Ploch M, Roots I. (1994). Pharmacokinetics of hypericin and pseudohypericin after oral intake of the hypericum perforatum extract LI 160 in healthy volunteers. J Geriatr Psychiatry Neurol. 7(suppl 1) S47-53. 

This herbal product has the most data available to support its usefulness as an antidepressant. Nevertheless, only minimal information is available about its pharmacology and its relative risk-benefit ratio. At least seven different biologically active chemicals have been isolated from crude extracts of hypericum. Several are ubiquitous in the plant kingdom. The exceptions are hypericin and pseudohypericin, which have been assumed to be responsible for any antidepressant activity of this product. Nevertheless, there is the potential for one or more of these seven compounds and their metabolites to mediate desired or undesired effects, particularly when used in combination with other medications (i.e., herb-drug interactions). 

The pharmacological activity of SJW extracts has recently been reviewed (55-58). Recent reports have shown that the antidepressant activity of Hypericum extracts can be attributed to the phloroglucinol derivative hyperforin (59-62), to the naphthodianthrones hypericin and pseudohypericin (18,63-65), and to several flavonoids (66-69). The role and the mechanisms of action of these different compounds are still a matter of debate. But, taking these previous findings together, it is likely that several constituents are responsible for the clinically observed antidepressant efficacy of SJW. 

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... 

A placebo-controlled, randomized clinical trial with monitoring of hypericin and pseudohypericin plasma concentrations was performed to evaluate the increase in dermal photosensitivity in humans after application of high doses of SJW extract (Table 2) (73). The study was divided into a single-dose and a multiple-dose part. In the single dose crossover study, each of the 13 volunteers received either placebo or 900, 1800, or 3600 mg of the SJW extract LI 160. Maximum total hypericin plasma concentrations were observed about four hours after dosage and were 0, 28, 61, and 159ng/mL, respectively. Pharmacokinetic parameters had a dose relationship that appeared to follow linear kinetics (73). 

LI 160 tablets containing 300mg extract (0.25mg hypericin and 0.52mg pseudohypericin per tablet). 

Animal studies Early pharmacokinetic studies in mice report that maximum plasma concentrations of hypericin and pseudohypericin were reached at six hours and were maintained for at least eight hours. The aqueous-ethanolic SJW extract used in this study contained 1.0 mg of hypericin (77). 

It was once thought that hypericin was the main active ingredient in St. John s wort. In 1994, it was reported that hypericin inhibited MAO-A (11). Further studies have shown that hypericin and pseudohypericin do not inhibit MAO-A, and hypericum extracts only inhibit MAO at extremely high concentrations (5). Furthermore, hypericin did not display a significant (>25%)... 

Two pharmacokinetic studies have examined the pharmacokinetics of hypericin and pseudohypericin (41,42). Standardized hypericum extract LI 160 (Jarsin 300 , Lichtwer Pharma GmbH, Berlin) was used in both trials. In Part I of the studies, subjects in both trials were administered a single dose of either 300, 900, or 1800 mg of the extract (one, three, or six coated tablets) at 10- to 14-day intervals. Each dose contained 250, 750, or 1500 p.g of hypericin and 526, 1578, or 3156 p,g of pseudohypericin, respectively. The doses were administered on an empty stomach in the morning after a 12-hour fast. Subjects fasted for an additional 2 hours after administration. Multiple plasma levels of hypericin and pseudohypericin were measured for up to 120 hours after administration. In addition, urine samples were collected in the study performed by Kerb and colleagues. After a 4-week washout from Part I, subjects were given one coated tablet containing 300 mg of hypericum extract three times a day (8 am, 1 pm, and 6 pm) before meals for 14 days. Blood samples were obtained over the 2-week dosing period. 

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 further 50 volunteers took 1800 mg/day of Hypericum extract LI 160 for 15 days. Sensitivity to SSI and UV-A light was measured before the first and 4 hours after the last drug intake. A marginal decrease in mean MED from 0.17 to 0.16 and a slight drop in mean MTD were observed. There was, however, no correlation between hypericin 1 and pseudohypericin 2 on the one hand and MED or MTD on the other hand. 

Due to their complex composition the pharmacokinetic assessment of herbal medications generally imposes serious technical and regulatory problems. As the active principle(s) of plant extracts are often not known it is difficult to decide which constituent(s) should actually be studied [218]. In the absence of a well defined therapeutically relevant chemical entity, characteristic constituents of herbal preparations are frequently employed for the purpose of standardization. Correspondingly, pharmacokinetic evaluations of Hypericum extracts have almost exclusively been based on the analysis of the naphthodianthrones hypericin 1 and pseudohypericin 2 which represent typical products of members of the genus Hypericum and are considered to be involved in some of their clinical effects. 

Table 3. Pharmacokinetic Parameters of Hypericin 1 and Pseudohypericin 2 After Oral Administration of 900 mg of a Methanolic Hypericum extract to Human Volunteers...

Figure 2. HPLC chromatograms ofextract of St. John s wort by (A) SFE with neat CO2 (40 C, 100 atm), and (B) ultrasonic extraction widi methanol. Peak I chlorogenic acid 2 rutin 3 quercitrin 4 quercetin 5 pseudohypericin 6 hypericin 7 hyperforin 8 adhyperforin.

It should first perhaps be mentioned that pressurized cold water has been successful in plant extraction. Hypericin, protohypericin, pseudohypericin and protopseudohypericin have been extracted from St. John s wort (Hypericum perforatum)... 

Figure 8 Thin-layer chromatography of Hypericum perforatum. The chromatograms show no important differences between the herbai drug and the extract except the red spots in front. Both spots are the resuit of chiorophyll, which is eliminated by the extraction procedure. Red spots Hypericin and pseudohypericin yeiiow and orange spots fiavonoids (glycosides R, 0.5 agly-cones R, 0.8) blue spot ubiquitous plant acids. Reference traces on the left Hypericin chlorogenic acid and quercetin. (Reproduced with permission from Beat Meier, Zeller AG, Herbal Remedies, Romanshorn, Switzerland.)...

Protopseudohypericin, pseudohypericin, proto pericin, and hypericin were extracted from Hypericum perforatum blossoms and baseline resolved on a C g column (A = 590nm) using a 12-min 70/30 -> 90/10 (at 8min hold 4min) (5/4 methanol/acetonitrile)/(wat [0.1 M triethylammonium acetate]) gradient [392]. Excellent peaks shapes were obtained. The total injected mass of the four compounds was i roximately 12pg. 

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... 

Figure 2 Typical negative ESI-MS mass spectrum of Hypericum perforatum extract. ESI-MS, electrospray ionization mass spectrometry Mi, rutin M2, hyperforin M3, pseudohypericin M4, hypericin M5, adhyperforin Me, isoquerci-trin/hyperoside. 

Hypericin and pseudohypericin (Fig. 21) are naphthodianthrones and occur in St. John s wort Hypericum perforatum, Clusiaceae). These dark-red compounds are accumulated in glands on the margin oiK perforatum leaves and petals. It was assumed for a long time that they represent the antidepressant principle in extracts of St. John s wort. Recently, however, it became evident that the prenylated acylphloroglucinols hyperforin and adhyperforin are responsible for most of the pharmacological effects. 

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