Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1999-04-16
2002-03-12
Geist, Gary (Department: 1623)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C514S025000, C514S783000
Reexamination Certificate
active
06355249
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the cost-effective recovery of saponins and sapogenins from plant material. In particular, the invention relates to the recovery of saponins and sapogenins from
Chenopodium quinoa
(Quinoa) (Chenopodiaceae) grain, bran and other plant parts in substantially pure and commercially useful forms.
2. Description of Related Art
The high levels of saponins found in certain plants has long been thought to be responsible for the medicinal effects of some of these plants (Waller, G. R. and K. Yamasaki,
Saponins used in Traditional and Modern Medicine,
Advances in Experimental Medicine and Biology, Vol.404, 1996, New York: Plenum Press). The presence of high levels of saponins in the seeds of plants such as Quinoa (
Chenopodium quinoa
) has restricted the use of the human consumption of this grain.
Quinoa originates from the Andes region of South America where it was a staple grain in pre-Spanish Conquest times. Traditional use declined after the Spanish Conquest (Galwey, N. W., et al.,
Food Sci. Nutr.,
42F:245, 1990) and cultivation and use of the grain was not widespread until a recent revival due to western interest in this crop as a high lysine, high protein grain for human consumption (De Bruin, A.,
J. Food Sci.,
26:872,1964). The principal obstacle to wider human consumption of this grain has and continues to be the bitter taste of the saponin content of the grain. These saponins have been shown to be anti-nutritive in animal studies (Gee, J. M., et al.,
J. Sci. Food Agric.,
63:201, 1993). In traditional use, the saponin content of the grain was reduced to acceptable levels by washing the grain in running water.
Since the revival of interest in Quinoa, a number of attempts have been made to devise practical methods to reduce the saponin content (Amaya-Farfan, J., et al., Removal of saponins from quinoa (
Chenopodium quinoa
Willd) grain by milling, 5th Int. Congr. Food Sci. Technol., Kyoto, Japan (1978); Gee, J. M., et al.,
J. Sci. Food Agric.,
63:201,1993; Reichert, R. D., et al.,
Cereal Chem.
63:471, 1986; Galwey, N. W., et al.,
Food Sci. Nutr.
42F:245, 1990; Rios, M. L. T., et al., Arch Latinoamer Nutr. 28:253, 1978; Ridout, C. L., et al.,
J. Sci. Food Agric.
54:165, 1991), including combinations of milling and washing. In all cases the saponin rich fraction was considered to be a waste product and was discarded.
The recent interest in nutraceuticals and the medicinal properties of plants has resulted in studies that have attributed the biological activity of many of these plants to their saponin content. Many interesting physiological and pharmacological effects have been attributed to saponins and/or the corresponding sapogenins including reduction of serum cholesterol (Price et al.,
CRC Crit. Rev. Food Sci. Nutr.
26:27 (1987)), inhibition of alcohol absorption (Yoshikawa, M. & J. Yamahara (1996),
In Saponins used in Traditional and Modern Medicine,
Edited by G. R. Waller and K. Yamasaki. pp.207-218. New York, Plenum Press, Vol. 404), inhibition of glucose absorption (Matsuda, H., et al.,
Biol. Pharmac. Bull.
20:717, 1997), facilitation of transdermal absorption and intestinal absorption of drugs (Gee, et al.
Toxic. in Vitro
3:85 (1989)), hypoglycaemic and anti-inflammatory effects (Honda, T., et al.,
Bioorganic Med. Chem. Lett.
7:1623,1997). Recently studies have shown that novel derivatives of the sapogenin oleanolic acid have potentially valuable pharmacological properties (Finlay, H. J., et al.,
Bioorganic Med. Chem. Lett.
7:1769,1997).
Saponins have been known to have adjuvant activity since the 1920's (Sjölander & Cox,
Adv. Drug Delivery Rev.
34:321 (1998)), and a significant body of research has been conducted to explore these properties, particularly with saponin containing extracts of
Quillaia saponaria.
Kensil (Kensil, et al.,
J. Immunol.
146:431(1991)) has demonstrated that a need exists for a substantially pure saponin that can be used as an adjuvant in relatively low quantities with low toxicity and side effects. Estrada et al. U.S. Pat. No. 5,688,772 teaches that all quinoa saponins obtained by water extraction are equivalent and active as immunological adjuvants.
In spite of this interest, only a very limited number of purified saponins and sapogenins are commercially available and practical procedures for large scale quantitative and qualitative recovery of highly purified saponins and sapogenins are lacking in spite of numerous publications describing analytical and laboratory scale procedures. The lack of suitable practical extraction and purification methods is also reflected in the relatively high cost of those compounds that are available.
Traditionally, the saponin content in plant extracts has been determined by bioassay or by GLC analysis of the sapogenins derived by hydrolysis of the saponins (Ridout et al.,
J. Sci. Food Agric.
54:165 (1991)). GLC analysis in particular requires extensive clean up and hydrolysis prior to derivatization and analysis and this is reflected in the prior art for the extraction and purification of these compounds. Recent developments in HPLC analysis in our laboratories have indicated that, in the case of quinoa saponins, the GLC approach of extensive purification does not give quantitative or qualitative recovery of the naturally occurring saponins.
The chemical nature of the saponins found in quinoa has been the subject of several investigations (Mizui, F., et al.
Chem. Pharm. Bull.,
38:375 (1990)); however, the procedures used in these investigations for the recovery of the saponins is not practical and applicable for commercial scale production. The studies of Mizui et al. (Mizui, F., et al.
Chem. Pharm. Bull.,
38:375 (1990)) and others have shown that the saponins found in quinoa are of the triterpene type.
The prior art for isolation of saponins from quinoa falls into two categories: a) an aqueous extraction route typically as described in Estrada et al., 1997, U.S. Pat. Nos. 5,597,807 and 5,688,772; and b) a more traditional hot alcohol solvent (Mizui, F., et al.
Chem. Pharm. Bull.,
36:1415 (1988); Mizui, F., et al.
Chem. Pharm. Bull.,
38:375 (1990)). Surprisingly, the inventors of the present invention, have determined that neither the aqueous extraction route nor the hot alcohol extraction route are particularly efficient in recovery of quinoa saponins from bran, nor do either solvent extract the saponins from quinoa seed or bran on a qualitative basis. Estrada et al. U.S. Pat. No. 5,688,772 teaches that water extracts of quinoa (10 g of hulls extracted by 2×100 mL of water) contain all or most of the saponins present in quinoa.
Surprisingly, while the aqueous extraction method provides an extract with similar saponin profiles to that now known to be present in quinoa grain or bran, the yield was only 20% of that obtained by the process of the present invention. Mizui et al. [Mizui, F., et al.
Chem. Pharm. Bull.,
36:1415(1988); Mizui, F., et al.
Chem. Pharm. Bull.,
38:375 (1990)] demonstrated that saponins could be extracted from quinoa bran with hot methanol with yields of between 20 and 25% depending upon whether a subsequent hot 50% methanol extract was employed. These yields are significantly less than those achieved using the process of the present invention.
Surprisingly, the inventors have also shown that extraction with pure alcohols is highly selective in that methanol preferentially extracts only one of the three main saponins.
Surprising also is the very low yield of saponins obtained by this approach which is reflected not only in the results achieved by the present invention, but also in the very low yield of saponins obtained by Mizui et al. (1.66%).
The prior art for the purification of quinoa saponins is lacking in any specific details that would allow commercial scale production of these compounds. For example, Estrada et al., 1997 U.S. Pat. Nos. 5,597,807 and 5,688,772, and Estrada et al. Conun.
Immun. Microbial & Infect. Dis.
21:2
Aubin Andrew A.
Ballantyne Krista
Muir Alister D.
Paton David
Geist Gary
Her Majesty the Queen in right of Canada as represented by the
Khare Devesh
LandOfFree
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