We have screened 7 separate botanicals all claimed to have immune enhancing activity. Adjuvant activity in our model was found for some, but not all extracts. Our most striking finding was the potency of the four Coriolous versicolor
extracts. These extracts are known to be rich in β-glucans, the presumed most active ingredient. While all four Coriolus extracts had activity over a wide range of doses, PSK supplied by ICM was the most active. In some experiments, this equaled or surpassed reactivity seen with an equal weight of purified β-glucan of yeast origin. The potency of PSK as an adjuvant was confirmed with a second conjugate vaccine as well, GD3-KLH. Since PSK contains less than 25% β-glucan polysaccharides by weight, this suggests that β-(1,4) backbone with β-(1,3) and β-(1,6) glucocytic linkages characteristic of Coriolous versicolor
β-glucans (as apposed to the β-(1,3) backbone with β-(1,6) linkages characteristic of yeast β-glucans) may have unique potency. This could also be a consequence of the protein core in the protein bound polysaccharides which characterize PSK and PSP[14
]. Both PSK and PSP have been described to have wide ranging impact on WBC count, phagocytic functions, T-helper cell activation, T-cell function and cytokine production when tested either in vitro
or administered in vivo
]. Neither have been used as immunological adjuvant mixed with vaccine and there has been no attempt to link particular structures in PSK or PSP with adjuvant activity. Our findings suggest the possibility that β-glucans in Coriolous extracts are uniquely potent as immunological adjuvants, a possibility we are pursuing initially by further fractionating PSK and testing the individual fractions. An initial attempt at fractionating PSK demonstrated that ethanol extraction, the method that worked well for astragalus, had no detectable impact on PSK.
We have identified saponins and in particular the saponin fraction QS-21 and the semisynthetic saponin mix GPI-0100 as uniquely potent immunological adjuvants when mixed with conjugate vaccines containing glycolipids or peptides chemically conjugated to keyhole limpet hemocyanin (KLH)[11
]. The maximal doses of QS-21 used in mice (20µg) and patients (100µg) were selected for minimal weight loss in mice and acceptable local erythema/induration and systemic flu-like symptoms in patients[18
]. The quest continues for immunological adjuvants with more potent adjuvant activity and more limited local and systemic toxicities. While most studies have been focused on Quillaja saponaria
saponins, there are many additional botanicals expressing other saponins. A possible case in point is Astragalus membranaceus
which we demonstrate here to have significant adjuvant activity in the 95% ethanol fraction.
is a well known traditional Chinese medicinal plant used widely for a variety of indications. The main constituents of the A. membranaceus
root are polysaccharides, saponins and flavonoids. The cyclolanostane-type saponins have been identified as the most active ingredient with significant lymphocyte proliferation and immunostimulatory activities[19
]. Using the hemolytic activity of saponins from various botanicals as a surrogate for toxicity, the hemolytic and immunological adjuvant activities of a series of saponin rich botanicals have been compared [20
]. Saponins were extracted using 70% ethanol, ether and n-butanol. Fifty, 100 and 200mcg had comparable activity, augmenting antibody titers against ovalbumin by approximately 10 fold. Saponins of A. membranaceus
were identified as having lower hemolytic activity and stronger adjuvant activity than saponins in the other botanicals tested. We demonstrate here that the 95% ethanol extract of A. membranaceus
saponins (the saponin rich fraction) also augments antibody responses against weak glycolipid autoantigens such as globo H and strong xenoantigens such as KLH. The 95% ethanol extract tested here consists of approximately 40% saponins. At least 15 such separate saponins have been identified in A. membranaceus
saponins and the chemical structures defined[21
]. Four of these (astragalosides I-IV) are commercially available. While purifying the other 11 individual saponins from extracted saponins for use as adjuvants would be difficult, the recent description of the total chemical synthesis of QS-21[22
] raises the possibility that these remaining individual A. membranaceus
saponins could be synthesized. The described low hemolytic activity of these saponins and potent adjuvant activity suggest that this would be fruitful. It is expected that even among this family of saponins some would have greater or lesser toxicity and that this might be distinct from their adjuvant activity.
It has been claimed by proponents of botanical medicine that crude or simple extracts of botanicals or mixtures of botanicals have unique potencies that can not be replicated or exceeded by any of the individual chemical constituents. Such a claim is a testable hypothesis, at least with regard to the saponins in the 95% ethanol extract of astragalus and β-glucans in PSK. This is especially relevant in the case of immunological adjuvants where efficacy frequently varies with dose but dose administered is limited by toxicity. The clear superiority of QS-21 over cruder fractions or unfractionated Quillaia saponaria saponins was largely a consequence of the superior immunogenicity/toxicity ratio. It may be that saponins are not the only immunologically active components in the 95% ethanol fraction or that a mixture of these saponins will prove superior to any one, but again this should be testable. The same applies to the unexpected potency of the β-glucans in PSK. In addition, if the goal is optimizing the adjuvant activity of botanicals, identification of the most active components is a necessary first step to developing relevant markers for improved methods of extraction and for confirming relevant batch to batch consistency. The studies described here are our first steps in these directions.