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MedicinalReprinted from: Perspectives on new crops and new uses. 1999.
J. Janick (ed.), ASHS Press, Alexandria, VA.
Immune Stimulants and Antiviral Botanicals:
Echinacea and Ginseng
Preparations of echinacea and ginseng likely comprise the most popular and commercially successful combination of medicinal plant products in North America. Interestingly, the development of Echinacea spe-cies for medicinal purposes stems from use by indigenous American people, but research has been conductedpredominantly in Germany (Bauer and Wagner 1991). On the other hand, the reputation of ginseng is firmlyplanted in China and has spread west in modern times, research in China, Japan, and Korea, being bolsteredby European investigations of mainly extracts of the Asian species, Panax ginseng. Following its discoveryoutside present-day Montreal, Quebec, in 1716 (Foster 1992), the trade in North American ginseng, Panaxquinquefolius, grew steadily and now constitutes a considerable market in the Orient, where roughly 90% ofCanada- and US-grown root is sold. Only one of the additionally recognized Panax species, namely Panaxnotoginseng (Sanchi or Tienchi ginseng), is of commercial significance, primarily in Asia, where it finds ap-plication as a hemostatic, cardiotonic, and anti-infective agent (Gao et al. 1996).
The traditional use of echinacea by native Americans as a topical anti-infective and snake-bite remedy, and to assuage coughs associated with colds, was presumably the impetus for investigation of its anti-micro-bial potential. Anti-bacterial, anti-fungal and anti-viral activities were subsequently assessed for a variety ofpreparations, the identity of the particular Echinacea species being still somewhat equivocal (Bauer and Wagner1991). Latterly, attention has concentrated on the plant’s application to the treatment of symptoms of cold andother influenza-like infections, the basis for its economic success; any effectiveness in such conditions is widelyattributed to effect on the immune system—potentiation or “stimulation” being generally regarded more par-ticularly as “immunomodulation” (Bauer and Wagner 1991). Any benefit deriving from treatment with echinaceapreparations is thought to be due to such stimulation of the immune system, rather than to any direct anti-viraleffect.
Ginseng (P. ginseng) has a traditional reputation as a tonic and drug of longevity in Asia, where it has also long been used to treat a variety of diseases, including cancer and diabetes, as well as various infections;in modern times, it has been characterized as an ‘adaptogen’ or anti-stress agent, exerting a non-specific nor-malizing effect on bodily functions. Ginsenosides, also termed panaxosides, are triterpene saponins regardedas the main active constituents of Panax species; hydrophilic bioactive polysaccharides are held responsiblefor anti-complementary and anti-tumor activities (Gao et al. 1991). However, while the attention of the herbalindustry has focused almost exclusively on ginsenosides, almost all investigations of the immunomodulatoryeffects of ginseng have involved crude extracts of P. ginseng (see later) and polysaccharide fractions of Panaxspecies (Yun et al. 1993; Gao et al. 1996). The results of one human study with a proprietary P. ginsengextract, support the claim of enhancement of immune response against influenza and colds, after vaccination(Scaglione et al. 1996).
The German Commission E (Blumenthal et al. 1998) approves the use of Echinacea purpurea herb as follows: (1) “Internally, as supportive therapy for colds and chronic infections of the respiratory tract andlower urinary tract,” and (2) “Externally, on poorly healing wounds and chronic ulcerations.” E. pallida isrecognized as “supportive therapy for influenza-like infections.” Investigation towards identification of the priniciple(s) responsible for the claimed immunomodulatory effects of echinacea preparations, and related effort to establish effective standardized therapeutic formula-tions have focused on five classes of echinacea constituents, namely, caffeic acid derivatives, alkylamides,‘polyacetylenes’ (ketoalkenes/ketoalkynes), glycoproteins, and polysaccharides (Bauer 1988).
Echinacoside, a caffeoylic popular for “standardization” but absent in appreciable quantity from roots of Echinacea purpurea, “has low anti-bacterial and anti-viral activity, and does not show immunostimulatoryeffects” (Beuscher et al. 1989; Bodinet and Beuscher 1991). Echinacoside is present in the roots of E.
angustifolia and E. pallida at levels between 0.3% and 1.7%, but the roots of the two species can be differenti-ated by the occurrence of 1,3-0-(cynarin) and 1,5-0-dicaffeoylquinic acids present only in angustifolia. Cichoricacid, which possesses phagocytosis stimulatory activity in vitro and in vivo, is present in E. purpurea, but inonly trace amounts in E. angustifolia roots; it is also apparently subject to ready enzymatic degradation in theplant matrix and its content in commercial preparations has been observed to be extremely variable (Bauer1998).
In echinacea, alkamides accumulate primarily in the roots and inflorescences. The highest alkamide content is found in E. angustifolia root, while E. pallida roots contain only trace amounts. Small quantities ofalkamides also occur in the expressed juice of E. purpurea. Alkylamide content also has been reported tovary widely in commercial products (Bauer 1998). However, it is important to note that the alkylamides of E.
purpurea and E. angustifolia differ in general chemical structure. All but one from E. purpurea have twodouble bonds in conjugation with the amide carbonyl group, whereas eight E. angustifolia alkamides haveonly one double bond in conjugation with the amide group; E. angustifolia and E. purpurea share five alkamideswith the diene structure conjugated with the amide grouping, including the prominent isomeric tetraene pair,readily observable at 260 nm, whereas the three most prominent monene alkamides of angustifolia are mostconveniently detected at 210 nm (Bauer and Wagner 1991).
The main lipophilic constituents of E. pallida roots are ketoalkenes and ketoalkynes, all with a carbonyl group at the C-2 position of the carbon chains. It is misleading to designate these prominent lipophilic con-stituents of E. pallida root as polyacetylenes, in differentiation from the corresponding alkamide constituentsof E. angustifolia (15 identified) and E. purpurea (11 identified), since many of these latter compounds haveconjugated diacetylenic (diyn-) functionality (8 so far identified). Only two ketodiynes have been identifiedin E. pallida roots (Bauer 1998).
The hydrophilic, highly polar constituents, notably polysaccharides and glycoproteins, are present most prominently in expressed juice of the plant, and in aqueous and hydroalcoholic extracts of the aerial parts.
Two polysaccharides have been isolated from an aqueous extract of the aerial parts of E. purpurea and shownto stimulate phagocytosis in vitro and in vivo and to enhance production of oxygen radicals by macrophagesin a dose-dependent way (Stimpel et al. 1984; Lohmann–Matthes and Wagner 1989). Other activities, such asanti-infective and antitumor, have also been noted (Bauer 1998). In a phase-I clinical trial, a polysaccharidefraction, isolated from E. purpurea tissue culture, caused an increased production of leukocytes, segmentedgranulocytes, and tumor necrosis factor alpha (TNFa). In addition, high-molecular weight proteins have beenisolated from roots of E. angustifolia and E. purpurea; purified extracts of a glycoprotein-polysaccharide com-plex were found to exhibit B-cell stimulating activity and to induce release of interleukin I, TNFa and IFNa, b in macrophages. E. angustifolia and E. purpurea roots are reported to contain similar levels of glycopro-teins, E. pallida less (Beuscher et al. 1995). It should be noted, however, that these activities of polysaccha-ride and glycoprotein-polysaccharide have been observed with isolated fractions of polar echinacea extracts:traditional ethanolic extracts likely contain insignificant amounts of polysaccharides, and Bauer and Wagner(1991) have stated that “.only low concentrations of polysaccharides are present in the expressed juice andthey do not have the same composition as those in the extracts of aerial parts.” In addition, polysaccharidesare expected, for reasons of stability, to be ineffective by oral administration.
While levels and profiles of caffeoylics (Beuscher et al. 1995) and alkylamides from E. angustifolia and E. purpurea, as well as ketoalkenes and ketoalkynes from E. pallida, may be obtained by HPLC procedureswith excellent resolution and a high level of sensitivity (Maffei Facino et al. 1995), glycoproteins are mostconveniently assessed by an ELISA method (Egert and Beuscher 1992) and polysaccharides by enzymatichydrolysis and determination of total fructose (L.D. Lawson pers. commun.).
The stimulation of macrophage activity earlier noted for the polar glycoprotein-polysaccharide comple- ment of echinacea preparations, is paralleled by apparently similar activity of their lipophilic component. Infact, the lipophilic fractions of alcoholic extracts from E. angustifolia and E. purpurea aerial parts and rootsshowed the greatest activity (Bauer et al. 1989; Jacobson 1967); such extracts of aerial parts exhibited an“immunomodulating effect on the phagocytoxic, metabolic, and bactericidal activities of peritoneal macroph-ages in mice” (Bauer 1998).
Attempts to assess the therapeutic potential of echinacea preparations have mainly focused on measure- ment of phagocytoxic capacity by the carbon-clearance-test; enhancement by a factor of 1.5 to 1.7 was foundin purified alkamide fractions from E. angustifolia and E. purpurea roots (Bauer et al. 1989). However, over-whelmingly the major constituent of the alkamide fraction of E. purpurea root, namely the dodecatetraenoicacid isobutylamide 10 E/Z isomeric pair, exhibited only weak activity, and, in the language of Bauer (1998),“the most effective constituent remains to be found.” In consideration of all of the above, Bauer properlyinfers that immunostimulant activity of echinacea extracts resides in various classes of echinacea constituents.
In acknowledgment of that reality, what remains to be appreciated is which of these constituents are respon-sible for the medicinal value observed in the limited number of clinical studies available to this point. Ismeasurement of phagocytoxic activity a sensible, reliable indicator of immunostimulant potential in terms ofeffectiveness in the treatment of symptoms of cold and flu, and abatement of respiratory and urinary infec-tions? At an even further level of reduction, is there any real benefit—beyond identity and quality control con- siderations—in monitoring levels of individual echinacea constituents? And which ones would one select?As far as total tare of the five designated classes of compounds: caffeoylics, alkamides, ketoalkenes/ketoalkynes,glycoproteins, and polysaccharides, their relative importance regarding therapeutic effect yet remains to beproperly assessed.
Cold. Treatment with the dose equivalent of 900 mg root per day of an alcoholic extract of E. purpurea stimulated a faster reduction of cold symptoms in patients compared to the placebo group, and to a grouptreated with the equivalent of 450 mg per day of root (Braunig et al. 1992). An E. purpurea root extract,combined with vitamin C, was also found effective in treating the common cold (Scaglione and Lund 1995).
A double-blind, placebo-controlled study was conducted with freshly-expressed juice of E. purpurea 108 vol-unteers with chronic upper respiratory tract infections (Schoneberger 1992). Compared to the placebo, theechinacea preparation reduced the number of infections, lengthened the time between infections, shortenedthe duration of the illness and lessened the severity of symptoms; patients with diminished immune responseseemed to benefit most from the treatment. (Curiously, the German Commission E does not recognize theusefulness of E. purpurea root in treating colds, while approving E. purpurea “fresh above-ground parts, har-vested at flowering time” as a supportive treatment for colds and chronic infections of the respiratory tract andlower urinary tract).
Infections. E. purpurea expressed juice promoted faster healing in bronchitis, a viral infection, than an antibiotic (Baetgen 1988) and, as adjuvant, reduced the frequency of occurrence of Candida infection betterthan econazol nitrate (Coeugniet and Kuhnast 1986).
A hydroalcoholic tincture of E. pallida roots produced significantly faster recovery from upper respira- tory tract infections than placebo (Braunig and Knick 1993). A placebo controlled, double-blind trial con-ducted on 160 adults (Dorn et al. 1997) indicated that a daily dose of 900 mg of the extract of E. pallida rootwas effective in shortening the duration of upper respiratory tract infections in infected adults, whether ofbacterial or viral origin; however, the precise character of the preparation cannot be ascertained from the pub-lished information.
Clearly, there is a need for more and better studies to determine the most effective and condition-appro- priate echinacea preparations, with respect to species, plant part, character of preparations, and recommendeddosage. Given the present state of knowledge of the pharmacology of echinacea constituents, attempts attherapeutic standardization based on individual chemical constituents are unrealistic—pending identification of the constituent(s) responsible for beneficial clinical effect. While such efforts continue, any meaningfulstandardization must rest with careful and extensive chemical profiling of proven efficacious medications,and/or relevant bioactivity correlation.
Undoubtedly because of the difficulty of confirming such diffuse and protracted actions as “tonic,” reju- venating, and “adaptogenic” effects, most modern research on Panax has been conducted on more specificinfluences, with more easily determined end-points. Such experiments have invariably utilized either grossextracts of one sort or another, fractions of extracts, or individual ginseng constituents isolated from them.
Various Extracts, Fractions and Isolated Principles
In an attempt to resolve conflicting reports of ginseng’s effect on the immune system, a 70% ethanolic extract of P. ginseng was subjected to a comprehensive testing battery capable of detecting subtle immunechanges in mice, whose immune function was suppressed by cyclophosphamide, a common chemotherapeuticagent (Kim et al. 1990). The observation that basal natural killer (NK) cell activity was stimulated, supportsan immunomodulatory property for ginseng. However, while subchronic exposure helped stimulate recoveryof NK function in cyclophosphamide—immunosuppressed mice, NK activity was not further stimulated inpolyinosinic-polycytidilic treated mice—and other immunological parameters examined, such as T and B cellresponses, were not affected.
Using a sensitive two-site enzyme immunoassay specific for human basic fibroblast growth factor (bFGF), “bFGF-like immunoreactivity” was detected in a P. ginseng extract prepared by methanol extraction, followedby aqueous buffer treatment which precipitated a water-insoluble fraction, centrifugation, and filtration. AbFGF-like molecule was identified (Takei et al. 1996).
The ethanol-insoluble fraction of an aqueous extract of P. ginseng, tested for immunomodulatory activ- ity, was found to induce proliferation of splenocytes and to generate activated killer cells in vitro (Yun et al.
1993); the activated killer cells killed both NK cell sensitive and insensitive tumor target cells without MHC-restriction. Activation of splenocytes was mediated through endogenously produced interleukin-2. Treatmentof young male mice with this ethanol-insoluble ginseng fraction significantly reduced lung tumor incidence,compared with a cohort treated only with tumor-inducing benzo [a] pyrene. Prior to treatment with 95%ethanol, the original aqueous extract was solubilized in methanol and the methanol-soluble fraction, whichexhibited cytotoxicity to splenocytes in vitro, was excluded.
Stimulated by the observation that several polysaccharides from P. ginseng exhibit the immunological effects of increasing serum IgG level and activating the reticuloendothelial system (RES), Japanese research-ers have isolated two acidic polysaccharides, named ginsenan PA and ginsenan PB, whose detailed structureswere determined (Tomoda et al. 1993). Both polysaccharides displayed similar RES, anti-complementary,and alkaline-phosphatase-inducing activity. In this study, P. ginseng root was extracted, in the traditional Chi-nese manner, with hot water, the polysaccharides precipitated with cetyltrimethylammonium bromide, extractedwith dilute sodium chloride solution and the free polysaccharides precipitated by the addition of ethanol; col-umn chromatography, followed by dialysis and then gel filtration, yielded ginsenans PA and PB.
Immunostimulating activity of Panax has been observed also in polysaccharidic material extracted from the leaves of P. ginseng (Sum et al. 1994). In order to estimate the potential of ginseng leaves which can beharvested annually, as compared to ginseng roots which usually require at least four years of maturation, theseresearchers compared the immune complex binding enhancing activity of water and alkaline-soluble polysac-charide fractions of root and leaves of P. ginseng. Binding of glucose oxidase-anti-glucose oxidase complexes(GAG) to macrophages was enhanced by crude polysaccharide fractions GL-2 and GR-3, from leaves and rootrespectively, GL-2 being the most active of all four fractions; the alkaline-soluble leaf extract GLA-2 showedno activity. A pure active polysaccharide GL-4IIb2 containing 33% uronic acid, was isolated. GAG bindingto macrophages by GLIIb2 increased in a dose-dependent fashion. The authors judge that their observations“. suggest that the carbohydrate moiety of GL-4IIb2 activated the mononuclear phagocytic system in vivothrough an increase of Fc receptor expression mediated by de novo synthesis of the receptor protein and re-sulted in enhanced immune complexes clearance.” Immunostimulating polysaccharides have also been isolated from P. notoginseng; (Sanchi or Tienchi gin- seng). A RES-active polysaccharide, sanchinan A (Ohtani et al. 1987), and four distinct homogeneous activepolysaccharide fractions (Gao et al. 1991), have been isolated from the roots of P. notoginseng; in the latterstudy, one fraction showed strong anti-complementary activity, two significantly induced the production ofinterferon-g in the presence of concanavalin-A, and all induced the production of TNFa, using human serumand antibody-sensitized sheep red blood cells.
One study with an isolated saponin from P. ginseng, ginsenoside Rg1, assessed its stimulatory effect on immune function of lymphocytes in the elderly (Liu et al. 1995). A reduced responsiveness of lymphocyteshas been associated with aging as well as a lower percentage of IL-2 receptor positive cells, compared withthe younger population. Rg1 was found to stimulate proliferation of lymphocytes and to increase the fluidityof lymphocyte membrane in the aged.
In the past two decades, a considerable number of studies have been conducted on the proprietary P. ginseng extract, G115, from Pharmaton SA, Lugano-Bioggio, Switzerland. The immunomodulatory activityof G115 was assessed in mice (Singh et al. 1984). Pretreatment with the ginseng extract led to an increase inthe number of antibody forming cells, as well as of the titers of circulating antibodies. Pretreatment withginseng extract also modulated the cell mediated immune response in test animals. Ginseng extract signifi-cantly elevated NK cell activity in test animals, as compared to control animals, and enhanced production ofinterferon of both the pH stable and labile types, induced by 6-MFA, an interferon inducer of fungal origin.
Subsequently, three controlled human studies, with G115 conducted in Italy, examined the immunomodulatory effects of the extract, in one case was comparing G115 with an aqueous extract of P.
ginseng. In a controlled single-blind study (Scaglione et al. 1994) placebo was compared to 100 mg of G115administered every 12 hours for 8 weeks to patients suffering from chronic bronchitis. The functioning ofalveolar macrophages, collected by bronchoalveolar lavage, was determined by measuring the phagocytic ac-tivity and killing power towards Candida albicans, which were “significantly . increased at the eighth weekof treatment.” The observed improvement in macrophage immune response was seen to indicate an importantrole for G115 in “the prevention and therapy of respiratory disorders (emphasis added).
A comparative double-blind study with healthy volunteers was conducted with G115 and an aqueous extract of P. ginseng root (PKD 167/79), using the same dosing regime followed in the previous study (Scaglioneet al. 1990). No details are provided for preparation of the aqueous extract, as are the mode of preparation andthe constituent profile of G115 not disclosed, except for a roughly 4% combined content in the seven majorginsenosides usually assayed for in ginseng materials. While both extracts demonstrated immunostimulatoryactivity, the two extracts differed in some parameters. A noticeable increase in phagocytosis was observed inthe fourth week in the group treated with G115, whereas elevation in the PKC 167/79 group “appeared to bedelayed to the end of the eighth week.” A similar difference was observed regarding increase in T helperlymphocytes (T4). These researchers interpreted “the earlier effective action of G115”—also evident in anenhancement of T4/T8 ratio—as due an earlier onset of immune response, as compared with PKC 167/79.
The final sentence of the publication: “It can be suggested that on the basis of its different actions on theimmune system, the G115 extract may have a different composition in comparison with the PKC 167/79 ex-tract in terms of ginsenoside content” (emphasis added).
The third human trial with G115 involved 227 volunteers at 3 private practices (Scaglione et al. 1996).
Subjects were given either placebo or 100 mg of G115 daily, for 12 weeks, an anti-influenza polyvalent vacci-nation being administered after the 4th week. The frequency of influenza or common cold between 4 and 12weeks was 42 cases in the placebo group, and only 15 in the G115 group, the difference being highly signifi-cant (p<0.001). NK cell activity was also significantly higher in the ginseng group after 8 weeks, as werespecific antibody titers. The researchers concluded that G115 can improve the immune response in humansand can protect against influenza and the common cold.
As with echinacea, the results of studies assessing the immunostimulatory effect of ginseng preparations cannot be confidently interpreted in terms of particular plant constituents. The relative contribution of ginsenoside, polysaccharide, or other ginseng components cannot be estimated and, therefore, inferences re-garding relative effectiveness of different extracts of ginseng remain entirely speculative. Estimation ofginsenoside effect, for example, may depend not only on the total content of the most prominent 6–8 neutralginsenosides, but also on their relative ratios and perhaps contributions from their more-than-20 other closelyrelated triterpene saponins. As Scaglione et al. (1990) have posited, contradictory results from studies on theeffect of ginseng extracts on the immune system “could arise from the difference in dose levels, compositionand duration of therapy” (emphasis added).
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September 2013- Version n°23 ANRS - AC 11: RESISTANCE GROUP GENOTYPE INTERPRETATION: NUCLEOSIDE AND NUCLEOTIDE REVERSE TRANSCRIPTASE INHIBITORS Mutations associated with resistance Mutations associated with « possible resistance » • T215Y/F • T215A/C/D/E/G/H/I/L/N/S/V [1, 2, 3, 4] • At least 3 mutations among: M41L, D67N, K70R, L210W, T215A/C/D/E/G/H/I/L/N