Eupatorium perfoliatum L.: Phytochemistry, traditional use and current applications

Journal of Ethnopharmacology 138 (2011) 641–651

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm

Review

Eupatorium perfoliatum L.: Phytochemistry, traditional use and current applications Andreas Hensel a,∗ , Mareike Maas a , Jandirk Sendker a , Matthias Lechtenberg a , Frank Petereit a , Alexandra Deters a , Thomas Schmidt a , Timo Stark b a b

University of Münster, Institute for Pharmaceutical Biology and Phytochemistry, Hittorfstraße 56, D-48149 Münster, Germany Technical University of München, Chair of Food Chemistry and Molecular Sensory Science, Lise-Meitner-Straße 34, D-85354 Freising, Germany

a r t i c l e

i n f o

Article history: Received 7 April 2011 Received in revised form 25 August 2011 Accepted 2 October 2011 Available online 6 October 2011 Keywords: Eupatorium perfoliatum L. Inflammation Phytochemistry Pharmacology Plasmodium Sesquiterpene lactones

a b s t r a c t Ethnopharmacological relevance: Eupatorium perfoliatum L. originates from North America, where it has been widely used since centuries by native Indians. Additionally extracts are used also in Europe as immunostimulating agent for treatment of fever and cold. The following review summarizes published data on phytochemistry, ethnopharmacological use, as well as clinical and preclinical data. Materials and methods: Literature survey was performed via SciFinder® on papers and patents and by systematic research in ethnopharmacological literature at various university libraries. Results: The phytochemical composition of Eupatorium perfoliatum is described in detail for volatile oil, caffeic acid derivatives, flavonoids, sesquiterpene lactones, tannins, polysaccharides. Methods for analytical quality control, as well as specification for relevant lead structures can be deduced from published batch analysis. Preclinical studies indicate anti-inflammatory effects of ethanolic extracts, which can be correlated on a molecular level to eupafolin and sesquiterpen lactones. Antiplasmodial, antioxidative and immunomodulating activities are additionally discussed. Clinical data on the use of Eupatorium perfoliatum do not meet modern GCP requirements, but do indicate positive tendencies for use of ethanolic extracts for treatment of common colds. Conclusion: While the postulated immunostimulating properties of Eupatorium perfoliatum have not been confirmed by in vitro data, animal-studies and in vitro experiments with plant extracts both indicate antiinflammatory effects beside antiplasmodial effect against Plasmodium falciparum. Such an antiinflammation caused by the ethanolic extracts can be correlated well with clinical symptoms related to diseases as common cold, rheumatism, athritis etc. These data also support the plausibility of the plant’s traditional use by the North American indigenous population and early European settlers. In principle quality aspects of the herbal material have to be affirmed by establishing modern pharmacopoeial control methods to guarantee constant and reliable quality. © 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3.

Ethnopharmacological relevance and current use of Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Botanical characterization of Eupatorium perfoliatum L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Macroscopic/sensoric description of Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Macroscopic and microscopic description of Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemical composition of herbal material from Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Volatile oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Flavonoids (structural features see Fig. 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: EtOAc, ethyl acetate; EtOH, ethanol; MeOH, methanol; PBMC, peripheral blood mononuclear cells; p.o., per oral application; s.c., subcutaneous application; STL, sesquiterpene lactone; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase. ∗ Corresponding author. Tel.: +49 251 8333380; fax: +49 251 8338341. E-mail address: [email protected] (A. Hensel). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.002

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4.

5. 6. 7.

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3.3. Sesquiterpene lactones (STLs, structural features see Fig. 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Triterpenes and sterols (structural features see Fig. 6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Caffeic acid derivatives (structural features see Fig. 6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Fatty acids and fatty alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Pyrrolizidine alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological activities of Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Immunological activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Antiinflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cytotoxic and antibacterial effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Antioxidative effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Antiplasmodial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical investigations on Eupatorium perfoliatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical methods for quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Ethnopharmacological relevance and current use of Eupatorium perfoliatum

is indexed in the German Homeopathic Pharmacopoeia (HAB, 2010).

Eupatorium perfoliatum L. originates from North America, where it has been widely used since centuries by native Indians. Additionally extracts are used also in Europe as immunostimulating agent for treatment of fever and cold. The following review summarizes published data on phytochemistry, ethnopharmacological use, as well as clinical and preclinical data and correlates in vitro activities with the respective plant compounds. Leaves and flowering parts of the plant “common boneset” have been extensively used within traditional medicine of North America’s native inhabitants against fever and as diaphoretic agent (Locock, 1990; Mass and Hensel, 2008). Most natives groups from North America did know about the medicinal use of boneset, and traditional use as cold remedy and against fever is documented for Cherokee (as antipyretic agent), Chippewa (also as antirheumatic agent), Delaware (as antipyretic agent), Iroquies (as cold remedy), Menominee (as antipyretic agent), Mohegan (as cold remedy and antipyretic), Nanticoke (as antipyretic agent), Seminole (as antipyretic agent), Shinnecock (as cold remedy) (Moerman, 1998). Based on this application the plant was also named as “feverwort”. Settlers floating from Europe in the 18th century into the new continent adapted the use of this herbal material, but extended the use to treat malaria, yellow fever, dengue fever, flu, and common colds. The use for malaria was very popular and Eupatorium perfoliatum was clearly propagated as a substitute for quinine (Hall, 1974). Additionally the use of Eupatorium perfoliatum against arthritis, rheumatism and gout is documented (Stammel, 1986). In the late 19th century the use of plant extracts from Eupatorium perfoliatum was very popular in the United States, but was mainly restricted to the treatment of common colds accompanied by fever and for the treatment of rheumatism. The plant material was so widely used that it has been monographed from 1820 to 1900 in the United States Pharmacopoeia USP and from 1910 to 1945 in the National Formulary NF in order to set officinal quality standards. Despite this, the plant has been introduced to Europe during the last decades, but lacks a very broad prevalence. It is interesting that products on the European market are only related to homeopathic use for treatment of common cold, flu, liver and biliary diseases associated with fever and rheumatism. An evaluation commission of the former German drug agency (Federal Office for Drugs and Medicinal products, commission D, 1985) published a monograph for the homeopathic use of Eupatorium perfoliatum, which allows officially the use in homeopathic preparations. Based on this and for quality assurance Eupatorium perfoliatum

2. Botanical characterization of Eupatorium perfoliatum L. Eupatorium perfoliatum L. (syn. Eupatorium connatum MICH., Eupatorium glandulosum MICH.), Asteraceae, tribe Eupatorieae, is also known as boneset, feverwort, ague weed, Indian sage or thoroughwort. The genus Eupatorium includes about 38 species, which are found mostly in East Asia and North America. In Europe only Eupatorium cannabinum, is found. Herbal material from Eupatorium perfoliatum for medicinal purposes is produced mainly in North America, but plant material is also cultivated more and more in Europe. For that reason pharmacognostic and analytical differentiation between Eupatorium cannabinum and Eupatorium perfoliatum has to be ensured. 2.1. Macroscopic/sensoric description of Eupatorium perfoliatum The perennial herbaceous plant (1–1.5 m) with big rhizomes can be found mostly in areas with wet soils. After grinding of the leaves a characteristic aromatic smell (volatile oil) can be realized, and also a bitter taste (sequiterpene lactones) gets obvious. Well developed, lanceolate leaves are densely woolly, glandular, medium-sized and deciduous; opposite standing leaves are typically stem-clasping and exicaul, which is related to the Latin name “perfoliatum”, meaning that the stem is growing “through the leaves”. 10–20 white tubular flowers with cylindrical involucres are aggregated in inflorescences forming corymbs. 5 brown or black anthers are fused together, the scars of the style are longer than the corolla. The fruit, formed from the inferior ovary, is a gard, dry achene with a typical pappus system. Detailed morpholocical characteristics can be found in Fig. 1. 2.2. Macroscopic and microscopic description of Eupatorium perfoliatum Dried herbal material (Fig. 2) is composed of white to slightly yellow stem parts with swammy ground tissue and slightly brownish bark on the outer side. Parts of the leaves are bright green and with woolly trichomes. Star-type white to white-yellowish tubular flowers (2–4 mm) can be identified easily. Fruits and pappi should be absent (the material should be harvested before fruiting takes place). In contrast to herbal material of Eupatorium perfoliatum the more fibrous herbal material from Eupatorium cannabinum is more dark with a higher degree of dark-green leaves, longer tricomes (up to 7 mm or even longer), dark brown stem bark parts and flowers being larger (5–8 mm) than those from Eupatorium perfoliatum.

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Fig. 1. Eupatorium perfoliatum L.: (A) flowering aerial parts, (B) typical stem-clasping leaves, (C) inflorescence.

Microscopic characteristics from Eupatorium perfoliatum are the occurrence of many typical long trichomes (200–800 ␮m), consisting of 5–15 cells (Fig. 3). Trichomes are found dominantly on the bottom side of the leaves. Additionally essential oil glands, typical for Asteraceae, are found abaxial of the leaves. The tubular flowers with the coalesced petals and short silky pappus and stigmata can be used for microscopic identification (Fig. 3).

3. Phytochemical composition of herbal material from Eupatorium perfoliatum 3.1. Volatile oil The literature on occurrence of volatile oil is not congruent. While Schimmel (1915) excludes the existence of volatile oil in the herbal material, Woerdenbag et al. (1992) described 0.05% volatile oil with ␤-gurjunen, ␤-caryophyllenoxid, limonen, linalool, borneol, bornylacetat, (iso)-eugenol, ␣-copaen, ␤-elemen, ar-curcumen, ␤-caryophyllen, humulen und ␦-cadinen. Recent investigations of Maas (2011) indicated the presence of 1.8 mL/kg volatile oil (related to the dried herbal material). GC–MS analysis and subsequent identification of peaks by means of respective linear retention indices and NIST library revealed E-anethol (16.5 area%), carvon (7.6%), linalool (4.0%), camphor (2.5%), selin-11-en4-␣-ol (5.5%) and caryophyllenoxide (3.8%) as major components. Summarizing these data the herbal material seems to contain only minor amounts of volatile oil, but with a non-consistent composition, which remains to be investigated by a broad batch-tobatch analysis in order to elucidate the influence of seed material, growth conditions, harvest time, etc. 3.2. Flavonoids (structural features see Fig. 4)

Fig. 2. Macroscopic details from herbal material from Eupatorium perfoliatum.

Main flavonoids described by Habtemariam (2008) and Wagner et al. (1972) indicated the presence of flavonols kaempferol 1, astragalin (kaempferol-3-O-␤-d-glucoside) 2, nicotiflorin (kaempferol-3-O-␤-d-rutinoside) 4, quercetin 5, hyperoside

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Fig. 3. Eupatorium perfoliatum: microscopic details for plant identification: (A) stem with unbranched, multi-cell, uniseriate trichomes; (B) multi-cell, uniseriate, trichome; (C) lower part of the leaves with trichomes and glandulae; (D) ovar: essential oil glands on outer surface; (E) tubular flower; (F) stigmata.

OH

HO

O R3

R2

R1 OH

Kaempferol Astragalin Trifolin Nicotiflorin Quercetin Isoquercitrin Hyperosid Rutin Hispidulin Eupafolin Patuletin

O

1 2 3 4 5 6 7 8 9 10 11

R1 OH O-β-D-glucose O-β-D-galactose O-β-D-rutinose OH O-β-D-glucose O-β-D-galactose O-β-D-rutinose H H OH

R2 H H H H H H H H OCH3 OCH3 OCH3

Fig. 4. Structural features of flavonoids from Eupatorium perfoliatum.

R3 H H H H OH OH OH OH H OH OH

A. Hensel et al. / Journal of Ethnopharmacology 138 (2011) 641–651

(quercetin-3-O-␤-d-galactoside) 7, and rutin (quercetin-3-O-␤ -rutinoside) 8. Maas et al. (2008) identified isoquercitrin (quercetin-3-O-␤-glucoside) 6 and trifolin (kaempferol-3-O-␤-dgalactoside) 3 from a crude methanol/water (70/30, v/v) extract. The presence of the methoxylated flavon eupatorin was postulated in older literature (Latin, 1880; Shamel, 1892), but an exact documentation of structure elucidation is missing in these reports. In contrast to that the absence of flavones in CHCl3 extracts from Eupatorium perfoliatum is described in later publications (Herz et al., 1972). Also in our recent investigations eupatorin has not been detected in any fraction. Recent investigations (Maas, 2011) additionally identified the methoxylated flavonoid aglycones hispidulin 9, eupafolin 10, and patuletin 11 from the methanol-soluble part of a CH2 Cl2 extract of Eupatorium perfoliatum herbal material. More detailed experiments performed by washing of fresh leaves with CHCl3 and LC–MS/MS analysis of the extract indicated hispidulin, eupafolin, patuletin and kaempferol as constituents of the epicuticular fraction (Maas et al., 2011b). From the point of inflammatory activity of the herbal material described in traditional medicine, it should be considered that eupafolin is known to exhibit anti-inflammatory effects, as shown by in vivo data (Pelzer et al., 1998; Williams et al., 1999; Clavin et al., 2007) mainly by inhibition of transcription factor NF-␬B (Clavin et al., 2007). Therefore it can be assumed that eupafolin-containing fractions from Eupatorium perfoliatum can contribute to the potential anti-inflammatory effect of the herbal material. 3.3. Sesquiterpene lactones (STLs, structural features see Fig. 5) The six STLs which have been reported so far in older literature are either guaianolides such as euperfolid 12 and 11,13␣dihydroeuperfolid 13 (Bohlmann and Grenz, 1977), eufoliatin 14 and eufoliatorin 15 (Herz et al., 1977) or germacranolides such as euperfolitin 16 and euperfolin 17 (Herz et al., 1977). Maas et al. (2011a,b) identified 5S,6R,7R,8R,11R-2-oxo-8tigloyloxyguaia-1(10),3-diene-6,12-olide-14-carboxylic acid 18, a natural product until now only described for Eupatorium perfoliatum. Additionally the respective C-14 carboxyl-reduced STLs with an aldehyde group 19 and a hydroxyl group at C-14 20 were detected. Also the 2,14-dioxo-8-tigloyloxyguai-3-ene-6,12olide 21 was identified, which apparently can be dimerized biosynthetically to an unusual dimeric guaianolide 22 with an interguaianolide linkage between C-14 and C-4. Compounds 17–22 represent a homologous group of closely related guainolides, with 18 and 19 being considered as oxidation products of 20, while 21 represents a more stable tautomeric form of 20 (Maas et al., 2011a,b). Additionally the occurrence of a germacranolid (3␣,14-dihydroxy-8␤-tigloyloxy-6␤H,7␣H,11␣Hgermacra-1(10)Z,4Z-dien-6,12-olid) 31 is described, a heliangolid which has not yet been described in plants. STLs with the special characteristics of an exocyclic methylene group are known to be compounds reacting by Michael addition with other bionucleophils (e.g. SH-groups of proteins). This leads to the formation of potential antigens from the STL haptens which again may induce allergic reactions. Also blocking of enzymes and other changes in biochemical and physiological processes are known for such STLs. The only STL with such an exocyclic methylene group detected in Eupatorium perfoliatum until now is euperfolid 12. It should be considered that many STLs with such structural features are known to exhibit anti-inflammatory effects (Hall et al., 1979; Czeczot, 2000), mainly by inhibition of transcription factor NF-␬B (Rüngeler et al., 1999). It can be assumed that also these STLs, especially euperfolid, from Eupatorium perfoliatum can contribute to the potential in vitro anti-inflammatory effect of the herbal material described below.

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3.4. Triterpenes and sterols (structural features see Fig. 6) According to Dominguez et al. (1974) the petroleum benzine extract of Eupatorium perfoliatum leaves contains ␣-amyrin, ␤-sitosterol, stigmasterol, 3␤-hydroxyurs-20-ene, 3␤-acetoxyurs20-ene, urs-20-en-3-one and 3␤-hydroxyursane. Additionally campesterol, ␤-amyrin, lupeol, taraxasterol and pseudotaraxasterol have been identified (Hooper and Chandler, 1984). Recent GC–MS studies by Maas (2011) were performed on fractions obtained from the methanol-soluble part of the CH2 Cl2 extract after further purification by FCPC and Sephadex LH-20 column chromatography. Hydroxylated or carboxylated compounds were identified after derivatisation to the respective trimethylsilyl derivatives (Ogunkoya, 1981; Bauer, 2002; Yang et al., 2009) by GC–MS and evaluation of the respective mass spectra in comparison with published data. In addition to the already described triterpenes, ␤-amyrinacetate (3-O-acetylolean-12-en-3␤-ol) 23, ␣-amyrinacetate (3-O-acetylurs-12-en-3␤-ol) 24, ␤-amyrenone (3-oxoolean-12-en) 25 and lupenone (lup-20(29)-en-on) 26 were identified. The already described presence of ␣- and ␤-amyrin, lupeol, taraxasterol, stigmasterol, campesterol, and ␤-sitosterol was affirmed. 3.5. Caffeic acid derivatives (structural features see Fig. 6) From the ethyl acetate soluble fractions of a MeOH/water extract six caffeic acid derivatives have been isolated and identified. Beside 5-caffeoylquinic acid (chlorogenic acid), 3-caffeoylquinic acid (neochlorogenic acid) and 3,5-dicaffeoylquinic acid, beside three depsides of caffeic acid with glucaric acids have been isolated, which up to now have only been found in Eupatorium perfoliatum: 2,5-dicaffeoylglucaric acid 27, 3,4-dicaffeoylglucaric acid 28 and 2,4- or 3,5-dicaffeoylglucaric acid 29 (stereochemistry still unclear) (Maas et al., 2008). It is interesting that for many Eupatorium species the occurrence of caffeoyl quinic acids has been described (Pagani and Romussi, 1967; Lang, 2001; Clavin et al., 2007), but only very recent investigations have revealed those compounds for Eupatorium perfoliatum. Recent investigations (Maas, 2011) revealed the presence of N-(E)-cinnamoyl-l-aspartic acid 30. This compound belongs to a homologous class of N-phenylpropenoylamino acids, with interesting pharmacological activities (Hensel et al., 2007; Niehues et al., 2010). 3.6. Fatty acids and fatty alcohols Recent GC–MS studies (Maas, 2011) were performed on fractions obtained from the methanol-soluble part of the CH2 Cl2 extract after further purification by FCPC and Sephadex LH-20 column chromatography. After silylation the homologous series of free saturated C16 , C18 , C20 , C22 , and C24 fatty acids were identified. At lower concentrations also C17 , C19 , C21 , and C23 fatty acids were found. Additionally oleic acid (C18:19c ), linoleic acid (C18:29c, 12c ) and ␣-linolenic acid (C18:39c, 12c, 15c ) were found. Concerning fatty alcohols free n-octadecanol, n-eicosanol, n-docosanol and ntetracosanol were found. 3.7. Polysaccharides Investigations on polymeric carbohydrates indicated the presence of 1.1% of cold-water soluble polysaccharides, obtained after extraction of the herbal material with water, ethanol-precipitation of the extract and dialysis of the resuspended polymers (MWCO 3.5 kDa) (Deters et al., 2004; Maas, 2011). Additionally, a fructan content of 1.3% (m/m) was determined by enzymatic assay from a hot-water extract. Although no detailed

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A. Hensel et al. / Journal of Ethnopharmacology 138 (2011) 641–651 O

O

R

O

O

O

HO

O

H

O

H

O

O HO

O

O

H

O O

O HO HOH C

O O

O

Euperfolin 17: R = H

Eufoliatin 14

Eufoliatorin 15

Euperfolitin 16: R = OH O

O O

O

OH O

O

O

O

H HO

HO

O

O

H HO

O

O

O

O

O

O

Euperfolid 12

3α,14-Dihydroxy-8β-tigloyloxy6βH,7αH,11αH-germacra-1(10)Z,4Zdien-6,12-olid 31

11,13α-Dihydroeuperfolid 13

O

HO

14

O

H

O

O

2 8

O

Otig

= tig

(Ox.)

H O

20

H O

O

19

O

O

HO O

(Ox.) Otig

HO

H

HO

O Otig

O

H

O O

O

-H2O

H

[20*] O

18

H

O

Otig HO

O

H H

O

H

O O Otig

Otig H

H

O

O

21

O

O

22

Fig. 5. STLs from Eupatorium perfoliatum and proposed biosynthetical relations during the formation of the dimeric guaianolides (Maas et al., 2011a,b).

A. Hensel et al. / Journal of Ethnopharmacology 138 (2011) 641–651

647

R R

H H

H

H O

H

H

H

O

O

H

H

O H

23: β-amyrinacetate R1 = CH3, R2 = H 24: α-amyrinacetate R1 = H, R2 = CH3

25: β-amyrone

26: luenone

O C1OOH H C2 R 2O

C3

H C4

OR1 H OR3

OH

O

27: R1 = R4 = caffeoyl, R2 = R3 = H 28: R2 = R3 = caffeoyl, R1 = R4 = H

OH

29: R1 = R3 = caffeoyl, R2 = R4 = H, or R2 = R4 = caffeoyl, R1 = R3 =

R = caffeoyl

H

H C5 OR4 C6OOH

O

COOH COOH

30: N-(E )cinnamoyl-L-aspartic acid

N H

Fig. 6. Structural features of triterpenes, phytosterols, dicaffeoylglucaric acid esters and N-phenyl-propenoyl amino acid amide from the aerial parts of Eupatorium perfoliatum.

linkage analysis was performed an inulin-like (␤(2 → 1)-fructosylfructose) can be assumed, which is chemotaxonomically typical for species from Asteraceae (methodology see Louis et al., 2010; Maas, 2011). From an alkaline extract (NaOH 0.5 mol/L) two 4-O-methylglucurono-␤(1 → 4)-xylanes with molecular weights of 40 kDa and >500 kDa were isolated by Vollmar et al. (1986). 3.8. Pyrrolizidine alkaloids Pyrrolizidine alkaloids with a potential hepatotoxic risk on human health are commonly found within the genus Eupatorium. It is highly interesting, that the occurrence of this substance group can be excluded for Eupatorium perfoliatum by different investigations (Söntgerath, 1988; Woerdenbag et al., 1992; Raffauf, 1996). Also in recent analytical and phytochemical investigations by Maas et al. (2011) no indications for pyrrolizidine alkaloids have been found. The absence of these compounds in Eupatorium perfoliatum was also one rationale for the German Drug Commission of Pharmacists, to indicate that the herbal material form Eupatorium perfoliatum can regared as safe for medicinal purposes (Arzneimittelkommission, 1990). 4. Pharmacological activities of Eupatorium perfoliatum Despite the well documented use in the European drug market, preclinical and clinical studies on Eupatorium perfoliatum are limited. 4.1. Immunological activity Two isolated polysaccharides (1 mg/mL), obtained by alkaline extraction with NaOH 0.5 mol/L and characterized as 4-O-methylglucurono-xylans increased the in vitro phagocytosis of human granulocytes by about 30% related to the untreated control groups (Wagner et al., 1985a). A similar result was obtained in a chemoluminiscent test, indicating a 25% stimulation at 100 ␮g/mL

concentration. The effect was more pronounced for the highmolecular weight polysaccharide. Intraperitoneal application of the polysaccharides (10 mg/kg body weight) to mice and investigation of macrophage phagocytosis activity with the in vivo carbon clearance test showed increased activity (Wagner et al., 1985a). Using in vitro macrophage chemoluminiscent and granulocyte tests, also the sesquiterpenlactone eufoliatin 14 was shown to increase the phagocytosis rate (Wagner et al., 1985a,b; Vollmar et al., 1986). Recent investigations (Maas et al., 2011a,b) did not show any in vitro stimulation of peripheral blood mononuclear cells (PBMC) and murine macrophages (cell line RAW 264.7) after treatment with Eupatorium perfoliatum extracts raw polysaccharides) of different polarities (MeOH–, EtOH– and CH2 Cl2 ) or the cold-water soluble polysaccharides. The test extracts or RPS did not influence NO-production in macrophages, which could be a hint for an alternative stimulation of non-phagocytic first-line defence by macrophages. From these data a direct influence of the extracts on PBMCs and macrophages can be excluded. These data are in contrast to the results described concerning the stimulating influence of polysaccharides on granulocytes and macrophages, as described above. It has to be kept in mind that the two polysaccharides described by Wagner et al. (1985a) have been isolated under strong alkaline conditions, which leads to the extraction of strongly cell wall associated polysaccharides. Especially glucuronoxylans are well known to be part of the strong cell wall architecture and to be not extractable under non-alkaline or non-chaotropic conditions. This means such cell wall polysaccharides should not be part of aqueous or ethanolic extracts as they are normally used for preparation of medicinal plant extracts. This discrepancy has to be kept in mind: Eupatorium perfoliatum may have macrophage-stimulating polysaccharides (if the test, documenting these effects have not been positive due to contamination of the isolated polysaccharides by LPS), but these polymers will not be part of the commercially or traditionally used extracts. However, it should be reevaluated

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A. Hensel et al. / Journal of Ethnopharmacology 138 (2011) 641–651

if the tested polysaccharides have been LPS-depleted prior to use, because LPS contamination, which occurs regularly during aqueous extraction, will lead to dramatic activation of macrophages. Also effects found for the isolated STL eufoliatin (Wagner et al., 1985a,b; Vollmar et al., 1986) have to be assessed in a quantitative way to ensure that active and stimulating amounts of this STL are indeed present in relevant concentrations in the traditionally used extracts, prepared by water or alcohol. From these in vitro data direct stimulation activities of Eupatorium perfoliatum extracts on the first barrier immune defence have not been approved. 4.2. Antiinflammation Based on the traditional use of Eupatorium perfoliatum extracts for fever or rheumatism anti-inflammatory effects have been described (Benoit et al., 1976) in rat paw edema after two s.c. applications of EtOH extract (100 mg/kg body weight). A 12% reduction of edema was found, which is assessed to be quite low at this high doses. An EtOH extract was prepared as mother tincture according to homeopathic regulations, and was investigated in vitro on human monocytes concerning the influence on induction of inflammatory cytokines (Nauert et al., 2006). Results confirmed a dose-dependent reduction of IL-1 and PGE2, while MMP-1 has not been influenced. Recent in vitro investigations (Maas et al., 2011b) of MeOH–, EtOH– and CH2 Cl2 extracts indicated anti-inflammatory activity against LPS-stimulated RAW 264.7 macrophages by inhibition of NO release (IC50 > 100, 89, 19 ␮g/mL resp.). Bioassay-guided fractionation revealed eupafolin 17 and the dimeric guaianolide 22 to have prominent NO inhibiting activity (IC50 6 resp. 16 ␮M). Anti-inflammatory activity was found on gene and protein level by significant down-regulation of cytokines CSF-3, IL-1␣, IL-1␤, and chemokines CCL2, CCL22 and CXCL10. Also TNF was downregulated moderately (−17%). Investigation of gene expression of LPS-stimulated macrophages under the influence of CH2 Cl2 extract by gene microarray technique (1070 genes) clearly indicated by geneontological evaluation that the extract leads to down regulation of cytokines, chemokines and surface receptors which are associated with inflammatory activity of macrophages (Maas et al., 2011b). These in vitro data validate the potent anti-inflammatory activity claimed during traditional use of Eupatorium perfoliatum extracts seems to be validated. For structure-activity relations we assume that STLs and eupafolin contribute mainly to the respective effects, and future investigations should concentrate on evaluation of the primary target of these compounds on cellular level. It may be assumed that an inhibition of NF-␬B transcription factor may be one of the major targets for these compounds from Eupatorium perfoliatum. 4.3. Cytotoxic and antibacterial effects EtOH extract from the dried leaves of Eupatorium perfoliatum (1 kg leaves soaked for 2 weeks with absolute EtOH, yield 21 g dried extract) was tested for cytotoxicity against different cancer cell lines (HeLa, EAhy 926, L929) (Habtemariam and Macpherson, 2000). IC50 was determined with 12–14 ␮g/mL, indicating cytotoxic effects. These effects were correlated by bio-assay guided fractionation to the respective unpolar subfractions (CH2 Cl2 , hexane, EtOAc), while the water-soluble compounds from the water phase did not show any cytotoxicity. Using the unfractionated ethanolic extract for antibacterial testing within the agar-diffusion assay weak antibacterial effects were found for quite high substance concentrations (>150 ␮g per plate) against gram-positive bacteria (Staphylococcus aureus,

Bacillus megaterium) while no activity was found against Escherichia coli and Pseudomonas aeruginosa (Habtemariam and Macpherson, 2000). Another preliminary study on potential cytotoxic effects of Eupatorium perfoliatum against cancer cell line Neuro-2a found tumoricide activity at very high concentrations (IC50 2.4 mg/mL) of an EtOH extract (Mazzio and Soliman, 2009). Recent investigations (Maas, 2011) on cytotoxicity of EtOH, MeOH and CH2 Cl2 extracts from Eupatorium perfoliatum against human keratinocytes (non-tumorigenic HaCaT cell line, an international accepted in vitro cell system for the systematic evaluation of cytotoxic agents) over 48 h incubation time. Only CH2 Cl2 extract exerts cell vitality reducing impact at concentrations of <100 ␮g/mL. All other extracts and subfractions (1–100 ␮g/mL) did not influence cell vitality. Additionally, the influence of EtOH, MeOH and CH2 Cl2 extracts from Eupatorium perfoliatum on the murine macrophages RAW 264.7 cell line after 48 h incubation time showed that only the CH2 Cl2 extract reduced cell vitality (MTT test) at 100 ␮g/mL (Maas, 2011). Lower concentrations (<20 ␮g/mL) did not affect cell physiology. Also EtOH and MeOH extract did not affect cell vitality at 1, 10 and 100 ␮g/mL. Summarizing these data significant cytotoxic activities of polar extracts from Eupatorium perfoliatum can be excluded. 4.4. Antioxidative effects Acivity-guided investigation (Habtemariam, 2008) of a EtOH extract from fresh leaves and subsequent fractionation into petroleum benzine, CH2 Cl2 , EtOAc, butanol and water phase, followed by SephadexLH-20 chromatography revealed major antioxidant properties (DPPH test) within the EtOAc and butanol phase that contain flavonoids and protocatechuic acid as dominant compounds. 4.5. Antiplasmodial activity Based on the traditional use of Eupatorium perfoliatum for malaria Lira-Salazar et al. (2006) investigated homeopathic preparations with the dilution C20 and LM6 on mice, infected with Plasmodium berghei. Treatment was performed over 9 days p.o. with 200 ␮L of the 1:25 diluted preparation. Chloroquin (5 mg/kg bodyweight) served as positive control. The C30 dilution reduced the infection rate by 61%, LM6 by 40% and chloroquin by 74% compared to the untreated control group. Eupatorium perfoliatum test solutions revealed an increase in shizonts. Recent studies (Maas et al., 2011a) indicated that the STLenriched CH2 Cl2 extract of aerial parts of Eupatorium perfoliatum L. exhibits significant antiprotozoal activity under in vitro conditions, especially against Plasmodium falciparum (IC50 2.7 ␮g/mL). The search for active compounds showed that the dimeric guaianolide 22 is the most active constituent against Plasmodium falciparum (IC50 2.0 ␮M) and was less cytotoxic against rat skeletal myoblasts (CC50 16.2 ␮M) which resulted in a selectivity index (IC50 /CC50 ratio) of about 8. STLs 18, 31 and the flavonoid eupafolin 10 revealed only very low activity and selectivity. No significant activity of the extract and the STLs was observed against Leishmania donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi (Maas et al., 2011a). 5. Clinical investigations on Eupatorium perfoliatum Only one clinical investigation describing positive effects of Eupatorium perfoliatum against common cold is documented as non-placebo controlled, open study with n = 53 patients with common cold (Gassinger et al., 1981). One group was treated with a

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Table 1 Batch analysis (HPLC) of 5 representative batches of herbal material of Eupatorium perfoliatum for development of analytical specification. Peak designation

Content [g/100 g], related to dried starting material Batch 1a

3-Caffeoylquinic acid 5-Caffoylquinic acid 2,4/3,5-Dicaffeoylglucaric acid 3,4-Dicaffeoylglucaric acid 2,5-Dicaffeoylglucaric acid 3,5-Dicaffeoylquinic acid 4,5-Dicaffeoylquinic acid Hyperoside Isoquercitrin Trifolin Astragalin Eupafolin

c d e

± ± ± ± ± ± ± ± ± ± ± ±

0.04% 0.10% 0.01% 0.04% 0.04% 0.18% 0.01% 0.02% 0.15% 0.02% 0.04% 0.04%

Batch 3c

Batch 4d

Batch 5e

0.43% 1.90% 0.03% 0.04% 0.01% 2.15% 0.12% 0.38% 0.82% 0.14% 0.17% 0.52%

0.04% 1.67% 0.07% 0.07% 0.03% 1.56% 0.11% 0.53% 0.83% 0.23% 0.23% 0.34%

0.06% 1.61% 0.08% 0.07% 0.04% 1.42% 0.13% 0.29% 0.43% 0.11% 0.09% 0.59%

0.02% 0.12% 0.02% 0.02% 0.01% 0.25% 0.04% 0.06% 0.07% 0.05% 0.04% 0.13%

Herbal material from agricultural farming at author’s laboratories botanical garden, mean values from determinations from July 2008 + 2009 + 2010. Herbal material from agricultural farming at botanical Garden Institute of Pharmaceutical Biology, Münster, July 2008. Herbal material from agricultural farming at botanical Garden Institute of Pharmaceutical Biology, Münster, July 2009. Commercially available sample (IPBPEP2010) from global wholesale trading (Cassella-med, Cologne, Germany). Commercially available sample (lot 3458) from global wholesale trading (A. Galke, Gittelde, Germany).

homeopathic preparation from Eupatorium perfoliatum (D2, meaning 1:100 dilution titer), the other group was treated with acetyl salicylic acid (3× 500 mg/day). The efficacy of the drugs was assessed after 1, 4, and 10 days. Neither subjective complaints nor laboratory parameters nor body temperature differed between the two groups. This non-controlled study has to be assessed as using a non-GCP protocol and can therefore deliver only very limited information on the clinical efficacy of Eupatorium perfoliatum. Further on two non-controlled, open clinical studies with an Eupatorium perfoliatum containing registered drug from the German market (Contramutan® , containing homeopathic preparations from Echinacea angustifolia, Atropa belladonna, Aconitum napellus and Eupatorium perfoliatum) on 100 infants with common cold indicated improvement of symptoms after 3 days (Bentley and Grünwald, 2006). Another open and non-controlled study with Contramutan® and treatment of 4.443 patients from different age groups suffering from common cold and infections of the upper respiratory system indicates that the symptoms have improved after 3 days (Tradler and Eckert, 2001). Both studies have to be assessed as using a non-GCP protocol and can deliver therefore no valuable information on the clinical efficacy. A study published by Elsässer-Beile et al. (1996) investigated the influence of a complex homeopathic preparation, registered as medicinal product on the German market and containing Eupatorium perfoliatum, Echinacea angustifolia and Thuja occidentalis, on lymphocyte activity of n = 35 patients after surgical removal of solid maligne cancers. 23 of these patients obtained the test remedies, 12 patients served as untreated control. Leucocytes were isolated during and after the therapy, cultivated and investigated concerning their ability for immunological response on IL-1␣, IL-1␤, IL-2, IL-6, TNF-␣ und IFN-␥ production after mitogen stimulation. No significant differences were found between verum group and untreated control group. Therefore it can be assessed that the treatment has no influence on the lymphocyte activity, a finding which is in correlation with the preclinical data described above.

6. Analytical methods for quality control The German Homeopathic Pharmacopoeia does not list specific chromatographic methods for the unambiguous identification of Eupatorium perfoliatum. In literature only one identification and fingerprinting method by zone capillary electrophoresis for separation and identification of hyperoside, isoquercitrin, 5caffeoylglucaric acid, 3-caffeoylquinic acid and 3,5-dicaffeoylquinic

acid is described, with a validation of the quantitative determination of isoquercitrin (Lechtenberg et al., 2009). For quality control of Eupatorium perfoliatum herbal material and drug preparations a validated HPLC method for the quantification of caffeic acid derivatives and flavonoids has been described (Maas, 2011). A typical chromatogram (Fig. 7) allows the identification of 7 caffeic acid derivatives and 5 flavonoids and can serve well as a suitable fingerprint system for unambiguous identity testing. Quantitation can be made easily by using the lead-peaks for chlorogenic acid and isoquercitrin as references for calculation of the caffeic acid and flavonoid derivatives, a methodology which was validated according to ICH-Guidelines (ICH, 1995,1997) concerning specificity, linearity, accuracy, and precison. Batch analysis of five representative samples indicated a good qualitative coincidence of the relevant lead compounds, but with differences concerning contents of the lead compounds (Table 1) (Maas, 2011). Especially commercial batches are partly assessed to lack quality, probably due to too long storage. These data can lead to a possible pharmacopoeial specification for the drug material, which could be stated as follows: chlorogenic acid > 1.5%,

1,4

2

1,2

absorption (AU)

a b

0.24 1.53 0.09 0.06 0.04 1.18 0.21 0.19 1.16 0.10 0.19 0.19

Batch 2b

1,0

6

0,8 0,6 0,4

8 1

0,2

I.S. 34

0,0 0

10

7

5

20

9 10 11

30

12

40

50

60

time (min) 1 2 3 4 5 6 9 7 8 10 11 12

3-Caffeoyl quinic acid (neochlorogenic acid) 5-Caffeoyl quinic acid (chlorogenic acid) 2,4/3,5-Dicaffeoyl glucaric acid 3,4-Dicaffeoyl glucaric acid 2,5-Dicaffeoylglucaric acid 3,5-Dicaffeoyl quinic acid 4,5-Dicaffeoyl quinic acid Hyperoside Isoquercitrin Trifolin Astragalin Eupafolin

Fig. 7. Typical HPLC chromatogram of a methanolic extract from Eupatorium perfoliatum and correlation of main peaks to relevant compounds. I.S.: internal standard (ferulic acid).

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3,5-dicaffeoyl quinic acid > 1.0%, isoquercitrin > 0.4%, hyperoside > 0.2%, eupafolin > 0.2%. On the other side it has to be kept in mind, that the quantification of these compounds are only analytical markers and not necessarily related to pharmacological effects. Using the validated HPLC method, the distribution of the lead compounds between the different parts of the aerial material of Eupatorium perfoliatum (flowers, leaves, stem, total herb) was investigated over different times of the spring/summer/autumn vegetation period of three years (Maas, 2011). In principle chlorogenic acid was the most prominent compound (about 2.5%) in the total herbal material with a more or less constant content over the time interval from May to September. Chlorogenic acid is mainly located in the leaves, and can only be found in traces in the stems. 3,5-Dicaffeoylquinic acid accounted for about 1.5% in the herbal material, showing decreasing contents in late summer to autumn. This compound accumulates mainly in the flowers (up to 3.5%), but can also be found in the leaves to a minor extent (about 1–1.5%). In general, dicaffeoylquinic acids seem to accumulate in the flower parts of the plant, while the mono-substituted caffeoylquinic acids are prominent compounds in the leaves. Stem material does not accumulate any caffeoylquinic acid derivatives. Dicaffeoylglucaric acid derivatives are nearly exclusively accumulated in the flowers, with maximum content in late June to July during flowering period. The three different derivatives of this glucaric acid derivatives account each for maximal 0.1% of the total herbal material. For flavonoid glycosides the major compound isoquercitrin mainly accumulates in leaves, with about 2% during the early vegetation period, but with steadily decreasing content (about 0.6% in autumn). In contrast to that, hyperoside (about 0.2% in the herbal material), trifolin (about 0.1%) and astragalin (about 0.15%) show very constant contents over the year. The flavonoid aglycon eupafolin, representing the lipophilic derivatives, accumulates in the leaves (about 0.1–0.2%) and does also show no great seasonal changes.

7. Conclusions While the postulated immunostimulating properties of Eupatorium perfoliatum could not be confirmed, animal-studies and in vitro experiments with plant preparations strongly indicate both an antiplasmodial effect against Plasmodium falciparum and an anti-inflammatory effect. Such an antiinflammation caused by the ethanolic extracts can be correlated well with clinical symptoms related to diseases as common cold, rheumatism, arthritis etc. These data also support the plausibility of the plant’s traditional use by the North American indigenous population and early European settlers. This is in contrast to the recent use of Eupatorium perfoliatum in Europe, which is restricted to the application of homeopathic preparations against common cold, though the dosage of these preparations is comparably high through the usage of mother tinctures. While the antiplasmodial and antiinflammatory properties of Eupatorium perfoliatum could mainly be linked to the sesquiterpenes by in vitro assays, the constituents that may account for the claimed efficacy of homeopathic preparations against common cold remain unclear. This raises the question about a reasonable quality control that links analytical results to bioactivity. In case of the homeopathic usage, a control method that guarantees constant and reliable quality has to be restricted to characteristic analytical markers, which should be monitored by a validated HPLC-method. This should allow the unambiguous phytochemical identification of the material and especially the differentiation against the pyrrolizidin-alkaloid-containing species of Eupatorium

cannabinum, which by morphological and spatial similarity may easily adulterate drug material of Eupatorium perfoliatum. However, the development of an analytical method for the quantification of relevant STL and especially of the antiplasmodial agent 22 is desirable in order to allow assessment of antiplasmodial and anti-inflammatory properties and to facilitate further research on the phytochemistry of this compound class including the circumstances that lead to dimerization of STL to derivatives with increased bioactivity. Although the postulated immunostimulating properties of Eupatorium perfoliatum have not been confirmed, the antiinflammatory effects can be seen as a verification of the traditional use against inflammatory diseases. In principle quality aspects of the herbal material have to be affirmed by establishing modern pharmacopoeial control methods to guarantee constant and reliable quality. References Arzneimittelkommission, 1990. 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