WO2024057068A1 - Method to control the content of mono and bidesmosidic saponins in yucca extracts - Google Patents
Method to control the content of mono and bidesmosidic saponins in yucca extracts Download PDFInfo
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- WO2024057068A1 WO2024057068A1 PCT/IB2022/058718 IB2022058718W WO2024057068A1 WO 2024057068 A1 WO2024057068 A1 WO 2024057068A1 IB 2022058718 W IB2022058718 W IB 2022058718W WO 2024057068 A1 WO2024057068 A1 WO 2024057068A1
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- WO
- WIPO (PCT)
- Prior art keywords
- yucca
- saponins
- juice extract
- monodesmosidic
- juice
- Prior art date
Links
- 235000012044 Yucca brevifolia Nutrition 0.000 title claims abstract description 128
- 239000001653 FEMA 3120 Substances 0.000 title claims abstract description 125
- 235000004552 Yucca aloifolia Nutrition 0.000 title claims abstract description 125
- 235000017049 Yucca glauca Nutrition 0.000 title claims abstract description 125
- 229930182490 saponin Natural products 0.000 title claims abstract description 104
- 150000007949 saponins Chemical class 0.000 title claims abstract description 104
- 235000017709 saponins Nutrition 0.000 title claims abstract description 104
- 239000000284 extract Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 65
- 244000295923 Yucca aloifolia Species 0.000 title 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 53
- 238000006243 chemical reaction Methods 0.000 claims abstract description 25
- 244000110633 Yucca schidigera Species 0.000 claims abstract description 22
- 238000009835 boiling Methods 0.000 claims abstract description 22
- 235000006012 Yucca schidigera Nutrition 0.000 claims abstract description 21
- 239000002028 Biomass Substances 0.000 claims abstract description 20
- 235000004584 Yucca mohavensis Nutrition 0.000 claims abstract description 19
- 239000013049 sediment Substances 0.000 claims abstract description 18
- 239000002243 precursor Substances 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 8
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- 244000116042 Yucca brevifolia Species 0.000 claims description 124
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 27
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 claims description 20
- 229910000278 bentonite Inorganic materials 0.000 claims description 18
- 239000000440 bentonite Substances 0.000 claims description 18
- 229930002600 steroidal saponin Natural products 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 229910001868 water Inorganic materials 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 230000008020 evaporation Effects 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 5
- 239000000568 immunological adjuvant Substances 0.000 claims description 4
- 230000003308 immunostimulating effect Effects 0.000 claims description 4
- 239000006174 pH buffer Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- XMIIGOLPHOKFCH-UHFFFAOYSA-N 3-phenylpropionic acid Chemical compound OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 3
- 240000003347 Yucca baccata Species 0.000 claims description 3
- 235000017782 Yucca baccata Nutrition 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- 235000011007 phosphoric acid Nutrition 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 29
- 239000006286 aqueous extract Substances 0.000 abstract description 9
- 239000012646 vaccine adjuvant Substances 0.000 abstract description 9
- 229940124931 vaccine adjuvant Drugs 0.000 abstract description 9
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 239000001397 quillaja saponaria molina bark Substances 0.000 abstract description 8
- 238000012545 processing Methods 0.000 abstract description 7
- 241001532059 Yucca Species 0.000 abstract 8
- 230000000087 stabilizing effect Effects 0.000 abstract 1
- 229940092782 bentonite Drugs 0.000 description 16
- 241000196324 Embryophyta Species 0.000 description 15
- 239000001726 jatropha manihot extract Substances 0.000 description 15
- 229940106668 yucca extract Drugs 0.000 description 15
- 238000003825 pressing Methods 0.000 description 13
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 239000003814 drug Substances 0.000 description 11
- 235000013305 food Nutrition 0.000 description 11
- 229940079593 drug Drugs 0.000 description 10
- 239000002671 adjuvant Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 238000000605 extraction Methods 0.000 description 9
- 108010058846 Ovalbumin Proteins 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 229940092253 ovalbumin Drugs 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 150000005856 steroid saponins Chemical class 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 230000000845 anti-microbial effect Effects 0.000 description 4
- 238000001125 extrusion Methods 0.000 description 4
- PIGTXFOGKFOFTO-FVFWYJKVSA-N (2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8R,8aR,12aS,14aR,14bR)-8a-carboxy-4-formyl-8-hydroxy-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound O([C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)C[C@@H](O)[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O PIGTXFOGKFOFTO-FVFWYJKVSA-N 0.000 description 3
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 3
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013373 food additive Nutrition 0.000 description 3
- 239000002778 food additive Substances 0.000 description 3
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 229930002534 steroid glycoside Natural products 0.000 description 3
- 150000008143 steroidal glycosides Chemical class 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241001454523 Quillaja saponaria Species 0.000 description 2
- 235000009001 Quillaja saponaria Nutrition 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 235000019728 animal nutrition Nutrition 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 230000000656 anti-yeast Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 238000002705 metabolomic analysis Methods 0.000 description 2
- 230000001431 metabolomic effect Effects 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000003032 phytopathogenic effect Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 229940081836 yucca schidigera extract Drugs 0.000 description 2
- INLFWQCRAJUDCR-IQVMEADQSA-N (1R,2S,4S,5'S,6R,7S,8R,9S,12S,13S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icosane-6,2'-oxane] Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CCCCC4CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@H](C)CO1 INLFWQCRAJUDCR-IQVMEADQSA-N 0.000 description 1
- 241000746976 Agavaceae Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- ONCZQWJXONKSMM-UHFFFAOYSA-N dialuminum;disodium;oxygen(2-);silicon(4+);hydrate Chemical compound O.[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[O-2].[Na+].[Na+].[Al+3].[Al+3].[Si+4].[Si+4].[Si+4].[Si+4] ONCZQWJXONKSMM-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000004088 foaming agent Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000009215 host defense mechanism Effects 0.000 description 1
- 231100000003 human carcinogen Toxicity 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 229940080314 sodium bentonite Drugs 0.000 description 1
- 229910000280 sodium bentonite Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
- A23L2/04—Extraction of juices
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
Definitions
- the present invention is related to the field of production of purified saponins and saponin extracts from plants, promoting or preventing the formation of sediments of less soluble saponins during the preparation of aqueous extracts. More particularly, the present invention is in the field of preparation of extracts of Yucca schidigera Roezl. containing monodesmosidic and bidesmosidc steroidal saponins. Upon operational adjustment, the disclosed invention allows either the isolation and purification of monodesmosidic saponins for their use as vaccine adjuvants, or the stabilization of the bidesmosidic saponins extracted during the processing of the biomass of yucca, rendering stable aqueous extracts.
- Saponins comprise a large and diverse family of high molecular weight glycosides, widely distributed in the plant kingdom (Faizal, A., & Geelen, D. 2013. Saponins and their role in biological processes in plants. Phytochemistry Reviews, 12, 877-893; Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press; Sparg, S. G., Light, M. E., & Staden, J. Van. 2004. Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219-243; Vincken, J. P., Heng, L., de Groot, A., &found, H. 2007.
- Saponins classification and occurrence in the plant kingdom. Phytochemistry, 68, 275-297). They have a remarkable structural diversity which derives from their sapogenin structures and the sugars attached to them. Saponins are classified regarding the structure of the hydrophobic core known as aglycone or sapogenin, as triterpene or steroid saponins. Regarding the number of hydrophilic carbohydrate chains bonded to the hydrophobic aglycone, saponins containing one, two or three carbohydrate chains bonded to the aglycone are classified as mono-, bi- or tri-desmosides, respectively. Depending on the source of saponins, the sapogenins may contain different functional groups (e.g.
- Saponins are normally very difficult to separate due to their occurrence in highly complex mixtures of components with subtle structural differences among them, and their polar and amphiphilic properties (Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press).
- their isolation and purification require substantial efforts by sequential chromatographic steps under normal and/or reversed phase conditions and/or ultra I nano filtration, depending on the target homogeneity, and degree of purity.
- These processes are time consuming and expensive, and in the case of chromatographic techniques -with or without support- they require the use of organic solvents which might be toxic and harmful to the environment. Therefore, there is need for greener alternatives for saponin isolation in the industry.
- Yucca schidigera Roezl. ex Ortgies (yucca) is a plant belonging to the Agavaceae family, native to the southwestern United States and northern Mexico. Yucca extracts are one of the major industrial sources of steroid saponins ( Figure 1, Tables 1 and 2), approved in the USA by the Association of American Feed Control Officials (AAFCO) and the Food and Drug Administration (FDA), for their use as additives for the manufacture of beverages, cosmetics, animal food and poultry (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W. Oleszek & A.
- the commercial yucca products can be divided in three main groups: 1 ) The juice or aqueous extract produced by cold pressing the stem and concentrated by evaporation to approximately 50% w/w solids; 2) the juice absorbed onto inert carriers (clays, etc.); and 3) the dried pulverized yucca trunks comprised of pure botanical material.
- Yucca derived products are widely used as an animal feed additive for the control of ammonia and other odorous compounds in the gut and feces (Wallace, R. J., Arthaud, L., & Newbold, C. J. 1994. Influence of Yucca schidigera extract on ruminal ammonia concentrations and ruminal microorganisms. Applied and Environmental Microbiology, 60, 1762-1767). Additionally, the aqueous extract -juice- of yucca is a preferred natural foaming agent for beverages for human consumption and for cosmetics and personal care products (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W.
- US2715122A includes a process for saponin extraction using benzene as organic solvent, which is a known human carcinogen, and therefore, a forbidden supply for the production of extracts intended as raw materials for the formulation of vaccine adjuvants in the pharmaceutical industry.
- the regulatory framework of agencies such as FDA or EMA is very tight: The FDA issued an alert in June 2022 to drug manufacturers to avoid the use of benzene in their manufacture process; when the use of benzene become unavoidable to produce a drug product with a significant therapeutic advance, then its levels should be restricted to 2 parts per million (ppm), unless otherwise justified (Food and Drug Administration (2022).
- Chinese patent application CN108837775 A discloses a method to obtain steroid saponins from yucca which includes several steps of filtration, concentration and resin cleaning; this document discloses a process which combine several unitary operations in line with processes that are described in the state of art so far. Additionally, using this process it is not possible to obtain different kind of stable products enriched in mono and/or bidesmosidic yucca saponins, according to the client’s and/or researcher’s necessities.
- the present invention is related to the field of production of highly purified saponins and stable extracts containing saponins from plants, either to promote, or to prevent the formation of sediments of less soluble saponins during the preparation of aqueous extracts containing steroidal saponins, for their use as immunological adjuvants.
- this technology can be used either to promote or prevent the sediment formation according to the following: a) Promoting the sediment formation, through a controlled conversion of the filtered aqueous extract I juice precursor, allows the purification of a highly purified fraction of monodesmosic saponins having low water solubility, which might be used as ingredients of vaccine adjuvants.
- the above purification can be done heating up the fresh yucca extract below the boiling point, ideally to a maximum of 88 °C to promote a controlled conversion of the yucca juice precursor, and the corresponding precipitation of monodesmosidic saponins, followed of the removal of water-soluble components, and one or more cycles -preferably 3 cycles- of washing with water the precipitated components, and centrifugation to obtain the final product.
- the technology disclosed in this application allows the purification of monodesmosidic saponins without using organic solvents, chromatographic techniques, ultra I nano I nor reverse osmosis membranes (Figure 9). b) Preventing the sediment formation: To overcome the above-mentioned difficulties during the preparation of Yucca schidigera Roezl.
- FIG. 1 Flow diagram of the currently employed process for the production of yucca extracts (Prior Art).
- Figure 6 Shows a HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after boiling during 3.0 h, followed by incubation at 35 °C during 44 h.
- Figure 7 HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after bentonite treatment, followed by incubation at 35 °C during 44 h.
- Figure 9 Flow diagram of the disclosed process for the production of highly purified monodesmosidic saponins.
- FIG. 11 Relative titers of total IgG against OVA (expressed as titers relative to the control without adjuvant) in female BALB/c mice immunized with PBS buffer containing ovalbumin (OVA, 50 pg) (Control) and the following adjuvants (25 pg per dose): Freund's Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD).
- FCA Freund's Complete Adjuvant
- purified quillaja saponin QS-21 >95% purity UHPLC-UV
- monodesmosidic yucca saponins 91.1% purity HPLC-ELSD
- Figure 12 Flow diagram of the disclosed process to produce yucca extracts including the step of boiling of the fresh yucca juice after cold pressing step.
- Figure 13 Flow diagram of the disclosed process to produce yucca extracts including the step of bentonite treatment of the fresh yucca juice after cold pressing step.
- Figures 14 Comparison of the lot of yucca juice prepared by the standard process ( Figure 2) (bottle 2) and the lot prepared according to the flow diagram shown in the Figure 12 (bottle 1 ), including the boiling step for 3 h after cold pressing step.
- Figure 15 Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the standard process described in the Figure 2.
- Figure 16 Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the disclosed process described in the Figure 12.
- the present application relates to a method to produce either: a) a highly purified monodesmosidic saponins as vaccine adjuvants, and b) stable of extracts of Yucca schidigera Roezl. (yucca) containing monodesmosidic and bidesmosidic saponins found in the biomass of the said plant.
- stable refers to extracts of yucca free of precipitation upon incubation at room temperature.
- yucca plant refers to plants of the genus Yucca comprising, including, but not limited to: Yucca schidigera Roezl. , Yucca brevifolia Engelm., Yucca baccata T orr.
- the term “monodesmosidic saponin” as used herein refers to steroidal glycosides of yucca substituted with one chain of oligosaccharides as shown in Tables 1 and 2 and Figure 1.
- bidesmosidic saponin refers to steroidal glycosides of yucca substituted with two chains of oligosaccharides as shown in the Tables 1 and 2 and Figure 1.
- fresh yucca biomass refers to the roots, stems, and leaves of the yucca plant.
- hydrated dried biomass refers to hydrated dried roots, stems, and leaves of the yucca plant.
- vaccine adjuvant refers to as any immunomodulatory component(s) formulated with antigens in a vaccine to promote and/or enhancing the host defense mechanism.
- the present invention provides a method to produce stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19° Bx and the process for the obtention of concentrated yucca extracts having a Brix grade between 45 - 55 °Bx, preferably 50 °Bx, comprising:
- the production of stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19 °Bx is achieved by the method comprising:
- the present method allows to control the content of mono and bidesmosidic saponins in yucca extracts, suitable for isolation of steroidal saponins with immunostimulant properties, comprising: a) crushing and extruding fragmented harvested fresh or hydrated yucca biomass to obtain a first yucca juice extract (first precursor) having 15 - 25 °Bx, preferably, 19 °Bx, such first yucca juice extract then is submitted to: a.1 ) heating up below the boiling point and at a maximum temperature of 88 °C by steps, in 1 -10 steps, but preferably in 2-3 steps to promote a controlled conversion and a precipitation of monodesmosidic saponins; or a.2) a one-step boiling at 100 °C, or a heating above 100 °C, preferably, by Ultra High Temperature processing (UHT), or a.3) a bentonite treatment, or a.4) performing sequentially step a.2) and step a.3)
- step a.1 the method further comprising submitting such heated first fresh or hydrated dried yucca juice extract to a controlled conversion to promote instability, preferably 30 - 40 °C, more preferably at 35 °C, and, further f) optionally, after step a.1 ) and either after steps b, c), or d), the method further comprises submitting the sediment obtained after filtering to a water washing and centrifugation step in
- the present invention is also related to a highly pure yucca monodesmosidic saponins precipitate having between 60-100% purity, preferably 70-99% purity, at HPLC-ELSD, which is useful. as immunological adjuvant.
- the advantages of the present invention include, without limitation, the production of purified monodesmosidic saponins useful as vaccine adjuvants, and stable extracts of Yucca schidigera Roezl. containing mono and bidesmosidic saponins as commercial chemical raw material.
- the method of the present invention allows the purification of monodesmosidic saponins useful as vaccine adjuvants, and could be applied without using organic solvents, chromatographic techniques, ultra/nano/nor reverse osmosis membranes as described in the state of art so far.
- the method of the present invention allows the large-scale production of stable yucca extracts containing mono and bidesmosidic saponins, which are described as follows.
- stable yucca extracts can be obtained by boiling the fresh or hydrated dried yucca juice or by bentonite treatment which remains stable even after controlled conversion (at 35 °C for 44 h). Boiling the fresh yucca juice (during 0.5 and 3.0 h) or treating it with bentonite after the extraction process, allowed a lower conversion rate of the bidesmosidic saponins into monodesmosic ones, and an equilibrated relative amount (conversion rate of 0 to 95%) of bidesmosidic and monodesmosic saponins contained in the extract, which is stable even after its controlled conversion (at 35 °C for 44 h).
- a highly purified fraction containing monodesmosidic saponins can be obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water.
- Such highly purified fraction containing monodesmosidic saponins (91.1 % purity HPLC-ELSD, yield 4.2% dry weight) ( Figures 9 and 10).
- Example 1 HPLC-ELSD analysis of steroidal saponins in extracts of yucca.
- the samples were separated on an octadecylsilane end-capped column (250 x 4.6 mm, 5
- the detection was performed on an evaporative light scattering detector (ELSD), set at 50 °C and an air set at 2.5 bar was used as nebulizer gas.
- ELSD evaporative light scattering detector
- a calibration curve was determined by injection of standards of purified yucca saponins.
- the method allowed the determination of total yucca saponins, and the relative quantitation of bi- and monodesmosidic components by HPLC- ELSD.
- Example 2 HPLC-HRMS analysis of steroidal saponins in extracts of yucca.
- the analysis of the samples was performed by RP-HPLC, employing the same chromatographic conditions described in the Example 1.
- the HPLC chromatograph was coupled to a High-Resolution Electrospray Ionization Mass Spectrometer (HRMS) Thermo Fisher Scientific Exactive Plus Orbitrap (Bremen, Germany) in the mass range 100 - 1500 Da (negative mode).
- the sheath, auxiliary and sweep nitrogen flow rate were set at 9, 2 and the capillary temperature at 320 °C.
- the S lens RF was set at 50 and 1 arbitrary units, respectively.
- Spray voltage was set at -5 kV and the heater temperature at 150 °C.
- Extracted compound chromatograms were processed employing XcaliburTM Software.
- the conversion rate of the bidesmosidic saponins into monodesmosidic ones by boiling it during 0.5 and 3.0 h, or by treating it with bentonite short after the extraction/extrusion process was about 35, 49 and 84% compared to the no treated samples (Tables 1 and 2, and Figures 1 , 3 - 7).
- Example 4 Laboratory scale preparation of a stabilized yucca extract (50 °Bx) having an equilibrated relative amount of bidesmosidic and monodesmosidic saponins.
- Example 5 Preparation of a highly purified fraction containing monodesmosidic saponins obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water.
- mice Female BALB/c mice were immunized with PBS buffer containing ovalbumin (OVA, 50 .g) (Control) and the following adjuvants (25 jig per dose): Freund's Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD).
- FCA Freund's Complete Adjuvant
- FCA purified quillaja saponin QS-21
- monodesmosidic yucca saponins 91.1% purity HPLC-ELSD
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Abstract
It is disclosed a method to control the mono and bidesmosidic saponin content in yucca extracts, useful in a process of purifying such monodesmosidic saponins from Yucca schidigera RoezL (yucca) as precursors of vaccine adjuvants, comprising i) crushing and extruding fragmented yucca biomass or extracting the same; ii) removing the insoluble fraction by filtration; iii) controlling the conversion of the filtered aqueous extract; iv) removing water-soluble components, and v) recovering the precipitate by centrifugation to obtain a highly purified fraction containing yucca monodesmosidic saponins. Also, it is disclosed a process to prevent the sediment formation when obtained the yucca extracts by stabilizing the bidesmosidic saponins, comprising i) pasteurizing the fresh yucca juice obtained after extruded by ultra-high temperature processing (UHT) and/or boiling; ii) clarifying the pasteurized yucca juice by adding a flocculant; iii) removing the insoluble components from the clarified yucca juice by filtration, and; iv) concentrating the aqueous extract.
Description
METHOD TO CONTROL THE CONTENT OF MONO AND BIDESMOSIDIC SAPONINS IN YUCCA EXTRACTS
FIELD OF THE INVENTION
The present invention is related to the field of production of purified saponins and saponin extracts from plants, promoting or preventing the formation of sediments of less soluble saponins during the preparation of aqueous extracts. More particularly, the present invention is in the field of preparation of extracts of Yucca schidigera Roezl. containing monodesmosidic and bidesmosidc steroidal saponins. Upon operational adjustment, the disclosed invention allows either the isolation and purification of monodesmosidic saponins for their use as vaccine adjuvants, or the stabilization of the bidesmosidic saponins extracted during the processing of the biomass of yucca, rendering stable aqueous extracts.
DESCRIPTION OF RELATED ART
Saponins comprise a large and diverse family of high molecular weight glycosides, widely distributed in the plant kingdom (Faizal, A., & Geelen, D. 2013. Saponins and their role in biological processes in plants. Phytochemistry Reviews, 12, 877-893; Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press; Sparg, S. G., Light, M. E., & Staden, J. Van. 2004. Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219-243; Vincken, J. P., Heng, L., de Groot, A., & Gruppen, H. 2007. Saponins, classification and occurrence in the plant kingdom. Phytochemistry, 68, 275-297). They have a remarkable structural diversity which derives from their sapogenin structures and the sugars attached to them. Saponins are classified regarding the structure of the hydrophobic core known as aglycone or sapogenin, as triterpene or steroid saponins. Regarding the number of hydrophilic carbohydrate chains bonded to the hydrophobic aglycone, saponins containing one, two or three carbohydrate chains bonded to the aglycone are classified as mono-, bi- or tri-desmosides, respectively. Depending on the source of saponins, the sapogenins may contain different functional groups (e.g. methyl, hydroxyl, aldehyde, carboxylic acid, etc.) and spatial configuration. Besides, the composition, conformation, linkage, substitution, and branching patterns of the sugars attached increase enormously the diversity of the saponins (Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press). Although in different grades, most saponins share the property of forming colloidal solutions and lower the surface tension in aqueous media, which distinguishes them from other glycosides. Nonetheless,
their structural diversity is reflected by the myriad of biological activities and known uses for this family of compounds (Guglu-Ustundag, 0., & Mazza, G. 2007. Saponins: Properties, applications and processing. Critical Reviews in Food Science and Nutrition, 47, 231-258; Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press), indicating the importance of their study and their specific analysis in commercial products.
Saponins are normally very difficult to separate due to their occurrence in highly complex mixtures of components with subtle structural differences among them, and their polar and amphiphilic properties (Hostettmann, K., & Marston, A. 1995. Saponins. Cambridge University Press). In general, their isolation and purification require substantial efforts by sequential chromatographic steps under normal and/or reversed phase conditions and/or ultra I nano filtration, depending on the target homogeneity, and degree of purity. These processes are time consuming and expensive, and in the case of chromatographic techniques -with or without support- they require the use of organic solvents which might be toxic and harmful to the environment. Therefore, there is need for greener alternatives for saponin isolation in the industry.
Yucca schidigera Roezl. ex Ortgies (yucca) is a plant belonging to the Agavaceae family, native to the southwestern United States and northern Mexico. Yucca extracts are one of the major industrial sources of steroid saponins (Figure 1, Tables 1 and 2), approved in the USA by the Association of American Feed Control Officials (AAFCO) and the Food and Drug Administration (FDA), for their use as additives for the manufacture of beverages, cosmetics, animal food and poultry (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W. Oleszek & A. Marston (Eds.), Saponins in Food, Feedstuffs and Medicinal Plants, pp. 241-254. Springer Netherlands; Tamura, Y., Miyakoshi, M., & Yamamoto, M. 2012. Application of saponin-containing plants in foods and cosmetics in alternative medicine. In H. Sakagami (Ed), Alternative Medicine, pp. 85-102. InTech. Croatia). Their saponins - particularly monodesmosidic saponins - also have potent antifungal activity against phytopathogenic fungi and yeast strains recognized as food contaminants (Chapagain, B. P., Wiesman, Z., & Tsror, L. 2007. In vitro study of the antifungal activity of saponin-rich extracts against prevalent phytopathogenic fungi. Industrial Crops and Products, 26, 109-1 15; Miyakoshi, M., Tamura, Y., Masuda, H., Mizutani, K., Tanaka, O., & Ikeda, T. 2000. Antiyeast steroidal saponins from Yucca schidigera (Mohave Yucca), a
new anti-food-deteriorating agent. Journal of Natural Products, 25, 332-338; Wulff, E. G., Zida, E., Torp, J., & Lund, O. S. (2012). Yucca schidigera extract: A potential biofungicide against seedborne pathogens of sorghum. Plant Pathology, 61 , 331 -338).
The commercial yucca products can be divided in three main groups: 1 ) The juice or aqueous extract produced by cold pressing the stem and concentrated by evaporation to approximately 50% w/w solids; 2) the juice absorbed onto inert carriers (clays, etc.); and 3) the dried pulverized yucca trunks comprised of pure botanical material.
Yucca derived products are widely used as an animal feed additive for the control of ammonia and other odorous compounds in the gut and feces (Wallace, R. J., Arthaud, L., & Newbold, C. J. 1994. Influence of Yucca schidigera extract on ruminal ammonia concentrations and ruminal microorganisms. Applied and Environmental Microbiology, 60, 1762-1767). Additionally, the aqueous extract -juice- of yucca is a preferred natural foaming agent for beverages for human consumption and for cosmetics and personal care products (Cheeke, P. 2000. Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. In W. Oleszek & A. Marston (Eds.), Saponins in Food, Feedstuffs and Medicinal Plants, pp. 241-254. Springer Netherlands}. A further additional application is its use as a food additive due to its antimicrobial activity (Miyakoshi, M., Tamura, Y., Masuda, H., Mizutani, K., Tanaka, O., & Ikeda, T. 2000. Antiyeast steroidal saponins from Yucca schidigera (Mohave Yucca), a new anti-food-deteriorating agent. Journal of Natural Products, 25, 332-338; Suzuki, R., Ohno, H., Murakami, T., Shirataki, Y., 2020. Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics. Journal of Natural Medicine. 74, 306-310). Up to now, there are no reports of yucca saponins used as vaccine adjuvants.
The conventional processing of yucca biomass (Figure 2), by the extrusion of the plant botanical material renders an unstable fresh yucca juice, which become unstable short after the cold pressing of the biomass, forming an abundant and insoluble material at the bottom of the receiving tank. In addition to the afore mentioned instability, and also due to the natural variability of the active compounds in plants, some properties of the yucca extracts, such as profiles of saponins and antimicrobial activity become variable across production lots (Sastre, F., Ferreira, F., & Pedreschi, F. 2017. A systematic approach for the chromatographic fractionation and purification of major steroid saponins in
commercial extracts of Yucca schidigera Roezl. Journal of Chromatography B, 1046, 235-242; Suzuki, R., Ohno, H., Murakami, T., Shirataki, Y., 2020. Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics. Journal of Natural Medicine. 74, 306-310).
Current methods to prevent the afore mentioned defects include the heating of the resultant suspension to 80 - 90 °C (below the boiling point), filtering and recovery of the filtered juice in a second tank. However, the formation of precipitates in the filtered juice is still observed after the above treatment. Prior art analysis regarding process and/or methods for saponins extraction from yucca are showed in US Patent Application 2715122A which discloses a method of extraction of yucca saponins suitable for production at industrial scale. Nevertheless, the evidence described in US2715122A includes a process for saponin extraction using benzene as organic solvent, which is a known human carcinogen, and therefore, a forbidden supply for the production of extracts intended as raw materials for the formulation of vaccine adjuvants in the pharmaceutical industry. In that regard, the regulatory framework of agencies such as FDA or EMA is very tight: The FDA issued an alert in June 2022 to drug manufacturers to avoid the use of benzene in their manufacture process; when the use of benzene become unavoidable to produce a drug product with a significant therapeutic advance, then its levels should be restricted to 2 parts per million (ppm), unless otherwise justified (Food and Drug Administration (2022). “FDA alerts drug manufacturers to the risk of benzene contamination in certain drugs”, https://www.fda.gov/drugs/pharmaceutical-quality- resources/fda-alerts-drug-manufacturers-risk-benzene-contamination-certain-drugs).
Chinese patent application CN108837775 A discloses a method to obtain steroid saponins from yucca which includes several steps of filtration, concentration and resin cleaning; this document discloses a process which combine several unitary operations in line with processes that are described in the state of art so far. Additionally, using this process it is not possible to obtain different kind of stable products enriched in mono and/or bidesmosidic yucca saponins, according to the client’s and/or researcher’s necessities.
This analysis of prior art shows that despite the extraction processes of yucca saponins have been described, these are composed by several steps, use organic solvents, or does not offer the possibility to obtain stable products enriched in mono and/or
bidesmodidic yucca saponins, respectively, which are oriented to different applications according to their composition, such as in the pharmaceutical industry.
SUMMARY OF THE INVENTION
The present invention is related to the field of production of highly purified saponins and stable extracts containing saponins from plants, either to promote, or to prevent the formation of sediments of less soluble saponins during the preparation of aqueous extracts containing steroidal saponins, for their use as immunological adjuvants.
As mentioned above, this technology can be used either to promote or prevent the sediment formation according to the following: a) Promoting the sediment formation, through a controlled conversion of the filtered aqueous extract I juice precursor, allows the purification of a highly purified fraction of monodesmosic saponins having low water solubility, which might be used as ingredients of vaccine adjuvants. The above purification can be done heating up the fresh yucca extract below the boiling point, ideally to a maximum of 88 °C to promote a controlled conversion of the yucca juice precursor, and the corresponding precipitation of monodesmosidic saponins, followed of the removal of water-soluble components, and one or more cycles -preferably 3 cycles- of washing with water the precipitated components, and centrifugation to obtain the final product. The technology disclosed in this application allows the purification of monodesmosidic saponins without using organic solvents, chromatographic techniques, ultra I nano I nor reverse osmosis membranes (Figure 9). b) Preventing the sediment formation: To overcome the above-mentioned difficulties during the preparation of Yucca schidigera Roezl. (yucca) aqueous extracts by boiling the fresh yucca juice coming from the cold press step (Figure 12) during 0.5 - 3.0 h before further processing. In an alternate embodiment, this objective is accomplished by a process which comprises the step of treating the fresh yucca juice coming from the cold press step (Figure 13) with bentonite before further processing. The goal of this object is to preserve the relative amount of bi- and monodesmosidic saponins contained in the extracts, which could also open an opportunity for their functionalization, and hence for the development of new products with different physicochemical and/or biological properties. The technology disclosed in this document also allows the large-scale production of stable yucca extracts containing mono and bidesmosidic saponins.
BRIEF DESCRIPTION OF THE DRAWINGS.
Figure 1. Chemical structure of mono- and bidesmosidic saponins previously reported in yucca, and their assignment in the HPLC-HRMS chromatograms.
Figure 2. Flow diagram of the currently employed process for the production of yucca extracts (Prior Art).
Figure 3. HPLC-ELSD chromatogram of the methanolic extract of a fresh botanical sample of yucca.
Figure 4. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after incubation at 35 °C during 44 h.
Figure 5. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after boiling during 0.5 h, followed by incubation at 35 °C during 44 h.
Figure 6. Shows a HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after boiling during 3.0 h, followed by incubation at 35 °C during 44 h.
Figure 7. HPLC-ELSD chromatogram of the yucca extract obtained by cold pressing after bentonite treatment, followed by incubation at 35 °C during 44 h.
Figure 8. HPLC-ELSD chromatogram of the stabilized yucca extract (50 °Bx) prepared by the laboratory scale process described in the Example 4.
Figure 9. Flow diagram of the disclosed process for the production of highly purified monodesmosidic saponins.
Figure 10. HPLC-ELSD chromatogram of a highly purified fraction (91.1 % purity HPLC- ELSD) containing monodesmosidic saponins obtained by centrifugation and washing with water -three cycles- the yucca juice (50 °Bx) sediment.
Figure 11. Relative titers of total IgG against OVA (expressed as titers relative to the control without adjuvant) in female BALB/c mice immunized with PBS buffer containing ovalbumin (OVA, 50 pg) (Control) and the following adjuvants (25 pg per dose): Freund's
Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD). On weekly basis, four doses of antigen/adjuvant were applied to each group of mice (immunized intraperitoneally; seven mice per condition). Three days after the fourth immunization, blood samples were recovered from each mouse for determination of the immunoglobulin titer by ELISA.
Figure 12. Flow diagram of the disclosed process to produce yucca extracts including the step of boiling of the fresh yucca juice after cold pressing step.
Figure 13. Flow diagram of the disclosed process to produce yucca extracts including the step of bentonite treatment of the fresh yucca juice after cold pressing step.
Figures 14. Comparison of the lot of yucca juice prepared by the standard process (Figure 2) (bottle 2) and the lot prepared according to the flow diagram shown in the Figure 12 (bottle 1 ), including the boiling step for 3 h after cold pressing step.
Figure 15. Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the standard process described in the Figure 2.
Figure 16. Shows a HPLC-ELSD chromatogram of the yucca extract prepared by the disclosed process described in the Figure 12.
DETAILED DESCRIPTION OF THE INVENTION.
As described earlier, the present application relates to a method to produce either: a) a highly purified monodesmosidic saponins as vaccine adjuvants, and b) stable of extracts of Yucca schidigera Roezl. (yucca) containing monodesmosidic and bidesmosidic saponins found in the biomass of the said plant.
The term “stable” as used herein refers to extracts of yucca free of precipitation upon incubation at room temperature.
The term “yucca plant” as used herein refers to plants of the genus Yucca comprising, including, but not limited to: Yucca schidigera Roezl. , Yucca brevifolia Engelm., Yucca baccata T orr.
The term “monodesmosidic saponin” as used herein refers to steroidal glycosides of yucca substituted with one chain of oligosaccharides as shown in Tables 1 and 2 and Figure 1.
The term “bidesmosidic saponin” as used herein refers to steroidal glycosides of yucca substituted with two chains of oligosaccharides as shown in the Tables 1 and 2 and Figure 1.
The term “fresh yucca biomass” as used herein refers to the roots, stems, and leaves of the yucca plant.
The term “hydrated dried biomass” as used herein refers to hydrated dried roots, stems, and leaves of the yucca plant.
The term “vaccine adjuvant” as used herein refers to as any immunomodulatory component(s) formulated with antigens in a vaccine to promote and/or enhancing the host defense mechanism.
The present invention provides a method to produce stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19° Bx and the process for the obtention of concentrated yucca extracts having a Brix grade between 45 - 55 °Bx, preferably 50 °Bx, comprising:
- harvesting fresh or hydrated dried yucca biomass,
- cutting the harvested fresh or hydrated yucca biomass in fragments having an appropriate size to be cold pressed,
- conventionally cold pressing the fragmented harvested fresh or hydrated yucca biomass,
- boiling the yucca extract resulting from cold pressing,
- filtering the boiled yucca extract, and
- concentrating the filtered yucca extract to a desired concentration.
In an alternate description, the production of stable yucca juice precursor having a Brix grade between 15 - 25 °Bx, preferably 19 °Bx, is achieved by the method comprising:
- harvesting fresh or hydrated dried yucca biomass.
- cutting the harvested fresh or hydrated dried yucca biomass in fragments as mentioned above.
- conventionally cold pressing the fragmented harvested fresh or hydrated yucca biomass as mentioned above.
- treating of the yucca extract resulting from cold pressing with bentonite, preferably, with an amount of 0 - 100 g/L bentonite suspension.
- filtering the bentonite-treated yucca extract.
- concentrating the filtered yucca extract to a desired concentration as mentioned above.
Specifically, the present method allows to control the content of mono and bidesmosidic saponins in yucca extracts, suitable for isolation of steroidal saponins with immunostimulant properties, comprising: a) crushing and extruding fragmented harvested fresh or hydrated yucca biomass to obtain a first yucca juice extract (first precursor) having 15 - 25 °Bx, preferably, 19 °Bx, such first yucca juice extract then is submitted to: a.1 ) heating up below the boiling point and at a maximum temperature of 88 °C by steps, in 1 -10 steps, but preferably in 2-3 steps to promote a controlled conversion and a precipitation of monodesmosidic saponins; or a.2) a one-step boiling at 100 °C, or a heating above 100 °C, preferably, by Ultra High Temperature processing (UHT), or a.3) a bentonite treatment, or a.4) performing sequentially step a.2) and step a.3) but cooling after step a.2) or vice versa, b) concentrating such first yucca juice extract obtained from step a) by either, conventional evaporation or membrane concentration techniques or a combination thereof, to obtain a second yucca juice extract (second precursor) having 25 - 35 °Bx, preferably 30 °Bx; c) filtering such second yucca juice extract after adjusted pH with a pH buffer, preferably such pH buffer is selected from H3PO4, HCI, citric acid, ascorbic acid or a mixture of two or more thereof. d) concentrating such filtered yucca juice extract, either by conventional evaporation, membrane concentration techniques or a combination thereof, to obtain a third yucca juice extract having 45 - 55 °Bx, preferably, 50 °Bx; and
e) pasteurizing at 88 °C, such third yucca juice extract to obtain a fourth yucca juice extract having 45 - 55 °Bx, preferably, 50 °Bx and a pH between 3.5 - 4.5, preferably, pH 3.9, and then optionally, drying to be stored, wherein after step a.1), the method further comprising submitting such heated first fresh or hydrated dried yucca juice extract to a controlled conversion to promote instability, preferably 30 - 40 °C, more preferably at 35 °C, and, further f) optionally, after step a.1 ) and either after steps b, c), or d), the method further comprises submitting the sediment obtained after filtering to a water washing and centrifugation step in 1 -5 cycles, preferably 2-3 cycles, and purified monodesmosidic saponins are separately recovered.
The present invention is also related to a highly pure yucca monodesmosidic saponins precipitate having between 60-100% purity, preferably 70-99% purity, at HPLC-ELSD, which is useful. as immunological adjuvant.
The advantages of the present invention include, without limitation, the production of purified monodesmosidic saponins useful as vaccine adjuvants, and stable extracts of Yucca schidigera Roezl. containing mono and bidesmosidic saponins as commercial chemical raw material. Thus, the method of the present invention allows the purification of monodesmosidic saponins useful as vaccine adjuvants, and could be applied without using organic solvents, chromatographic techniques, ultra/nano/nor reverse osmosis membranes as described in the state of art so far. Besides, the method of the present invention allows the large-scale production of stable yucca extracts containing mono and bidesmosidic saponins, which are described as follows.
Thus, stable yucca extracts can be obtained by boiling the fresh or hydrated dried yucca juice or by bentonite treatment which remains stable even after controlled conversion (at 35 °C for 44 h). Boiling the fresh yucca juice (during 0.5 and 3.0 h) or treating it with bentonite after the extraction process, allowed a lower conversion rate of the bidesmosidic saponins into monodesmosic ones, and an equilibrated relative amount (conversion rate of 0 to 95%) of bidesmosidic and monodesmosic saponins contained in the extract, which is stable even after its controlled conversion (at 35 °C for 44 h). On the other hand, after a controlled conversion of the untreated fresh extract (at 35 °C for 44 h), bidesmosidic saponins are almost completely converted into monodesmosidic ones (groups 1 - 3, see Table 2 below), being the dominant components of the sediment,
particularly saponins belonging to group 1 , see Table 2 below. For the untreated fresh extract, a conversion rate of the bidesmosidic saponins into monodesmosidic ones of 99% was achieved at 35 °C for 44 h. On the other hand, a conversion rate of 35, 49 and 84% was achieved by boiling (0.5 and 3.0 h) or by a bentonite treatment after the extraction/extrusion process (see Tables 1 and 2, and Figures 1 , 3 - 7).
A highly purified fraction containing monodesmosidic saponins can be obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water. Such highly purified fraction containing monodesmosidic saponins (91.1 % purity HPLC-ELSD, yield 4.2% dry weight) (Figures 9 and 10). After tested sixteen different yucca juice lots, purities (HPLC-ELSD) and yields (dry weight) of the purified monodesmosidic saponins of 78 - 99% and 1 - 14%, respectively, were achieved.
The invention disclosed in this document may be further understood by reference based on the following Examples, which are not intended to be limiting of the scope of the invention.
Example 1. HPLC-ELSD analysis of steroidal saponins in extracts of yucca.
The analysis of the samples was performed by Reversed Phase High Performance Liquid Chromatography (RP-HPLC) according to Sastre, F., Ferreira, F., & Pedreschi, F. 2016, “MALDI-TOF mass spectrometry and reversed-phase HPLC-ELSD chromatography for structural and quantitative studies of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Pharmaceutical and Biomedical Analysis, 120, 270- 282. The samples were separated on an octadecylsilane end-capped column (250 x 4.6 mm, 5 |im particle size) maintained at 40 °C, eluted with a linear gradient from 68% MeOH in H2O to 88% MeOH in 30 min and a flow rate of 1 mL/min. The detection was performed on an evaporative light scattering detector (ELSD), set at 50 °C and an air set at 2.5 bar was used as nebulizer gas.
For quantification purposes, a calibration curve was determined by injection of standards of purified yucca saponins. The method allowed the determination of total yucca saponins, and the relative quantitation of bi- and monodesmosidic components by HPLC- ELSD.
Example 2. HPLC-HRMS analysis of steroidal saponins in extracts of yucca.
The analysis of the samples was performed by RP-HPLC, employing the same chromatographic conditions described in the Example 1. The HPLC chromatograph was coupled to a High-Resolution Electrospray Ionization Mass Spectrometer (HRMS) Thermo Fisher Scientific Exactive Plus Orbitrap (Bremen, Germany) in the mass range 100 - 1500 Da (negative mode). The sheath, auxiliary and sweep nitrogen flow rate were set at 9, 2 and the capillary temperature at 320 °C. The S lens RF was set at 50 and 1 arbitrary units, respectively. Spray voltage was set at -5 kV and the heater temperature at 150 °C. Extracted compound chromatograms were processed employing Xcalibur™ Software.
The identification of 12 saponins in the samples was performed by spiking with reference standards and confirmed with highly accurate mass measurements (compounds N°: 9 - 12, 14 - 17, and 21 - 24, Tables 1 and 2, and Figure 2). This information, along with reference data from previous literature (Kowalczyk, M., Pecio, L, Stochmal, A., & Oleszek, W. 2011 , “Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl”. Journal of Agricultural and Food Chemistry, 59(15), 8058-8064; Sastre, F., Ferreira, F., & Pedreschi, F. 2016, “MALDI- TOF mass spectrometry and reversed-phase HPLC-ELSD chromatography for structural and quantitative studies of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Pharmaceutical and Biomedical Analysis, 120, 270-282 Sastre, F., Ferreira, F., & Pedreschi, F. 2017, A systematic approach for the chromatographic fractionation and purification of major steroid saponins in commercial extracts of Yucca schidigera Roezl”. Journal of Chromatography B, 1046, 235-242} allowed the tentative identification of other 14 components, previously reported in yucca (compounds N°: 1 - 8, 13, 18 - 20, 25 and 26; Tables 1, and 2 and Figure 1).
Table 1. Compounds identified in yucca by High-Pressure Liquid Chromatography coupled to High Resolution Electrospray Ionization Mass Spectrometry (HPLC-HRMS).
10leszek, W., Sitek, M., Stochmal, A., Piacente, S., Pizza, C., & Cheeke, P. (2001 ). Steroidal saponins of Yucca schidigera Roezl. Journal of Agricultural and Food Chemistry, 49, 4392-4396.
2 Kowalczyk, M., Pecio, L., Stochmal, A., & Oleszek, W. (2011 ). Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl. Journal of Agricultural and Food Chemistry, 59(15), 8058-8064,
Table 2. Structural diversity of yucca saponins. monomosidic saponins identified (groups 1, 2 and 3), previously reported in yucca
bidesmosidic saponins previously reported in yucca, tentatively identified by HPLC-HRMS
Example 3. Testing of the stability of yucca extracts obtained by aqueous extraction yucca biomass at laboratory scale.
We tested the stability of yucca extracts obtained under controlled laboratory conditions, by boiling the fresh yucca juice (during 0.5 and 3.0 h), or by bentonite treatment, and subjecting these samples to a controlled conversion (at 35 °C for 44 h) in parallel with the untreated fresh sample. As shown in Figures 2, 3 and 4, after a controlled conversion of the untreated fresh extract (at 35 °C for 44 h), bidesmosidic saponins are completely converted into monodesmosidic ones (groups 1 - 3), being the latter also the dominant components of the sediment, particularly saponins belonging to group 1 . Boiling the fresh yucca juice (during 0.5 and 3.0 h), (Figures 5 and 6) or by treating it with bentonite (Volclay® KWK Food Grade, low dust granular sodium bentonite with an average particle size between 20 and 70 mesh) (Figure 7) short after the extraction process, allowed to lower the conversion rate of the bidesmosidic saponins into monodesmosic ones. These
processes disclosed (boiling or bentonite treatment), allows to obtain an equilibrated relative amount (conversion rate of 0 to 95%) of bidesmosidic and monodesmosic saponins contained in the extract, which is stable even after its controlled conversion (at 35 °C for 44 h), suggesting that the amount of sediment can be controlled and mitigated. In particular, the conversion rate of the bidesmosidic saponins into monodesmosidic ones for the analyzed intact untreated fresh sample after a controlled conversion at 35 °C for 44 h was 99%. For the same sample, the conversion rate of the bidesmosidic saponins into monodesmosidic ones by boiling it during 0.5 and 3.0 h, or by treating it with bentonite short after the extraction/extrusion process, was about 35, 49 and 84% compared to the no treated samples (Tables 1 and 2, and Figures 1 , 3 - 7).
Example 4: Laboratory scale preparation of a stabilized yucca extract (50 °Bx) having an equilibrated relative amount of bidesmosidic and monodesmosidic saponins.
Y. schidigera Roezl. (yucca) powdered biomass (50 g) was extracted in deionized water (500 mL) for 15 min at room temperature using a magnetic stirrer. Then, the extract was centrifuged at 3220 RCF for 10 min (20 °C), and the supernatant was transferred to a bottle (1 L). The supernatant was then adjusted to pH = 3.8 with diluted HCI and clarified with bentonite (Volclay KWK Food Grade, 3 g/L) for 1 h using a magnetic stirrer. The extract was then filtered through paper (qualitative grade 2) and concentrated by evaporation in an oven set at 88 °C. After this process, a stabilized yucca juice (30 g) at 50 °Bx, 5.2% total yucca saponins, pH = 3.2, having a conversion rate of the bidesmosidic saponins into monodesmosidic ones of about 66% compared to the intact sample, was obtained (Figures 8 and 13).
Example 5. Preparation of a highly purified fraction containing monodesmosidic saponins obtained by centrifugation and washing of a yucca juice (50 °Bx) sediment with water.
An aliquot of a yucca juice (12 mL, 50 °Bx) was centrifuged at 3100 RCF for 30 min at 20 °C. The sediment was washed 3 times with mili-Q water (12 mL x 3) and centrifuged as previously described. After this step, the sediment was dried in an oven for 24 h at 50 °C, weighted, and analyzed by HPLC-ELSD and HPLC-HRMS as previously described in the Examples 1 and 2, respectively. This process allowed to obtain a highly purified fraction containing monodesmosidic saponins (91 .1% purity HPLC-ELSD, yield 4.2% dry weight) (Figures 9 and 10).
This process was tested in sixteen different yucca juice lots and the purities (HPLC-ELSD) and yields (dry weight) of the purified monodesmosidic saponins ranged from 78 - 99% and 1 - 14%, respectively.
Example 6. Pilot production testing of yucca extracts.
The results obtained after the preparation of an experimental lot of yucca juice by adding a boiling step for 3 h short after the extrusion process, as described in Figure 12, were consistent with the laboratory study (Example 3). Both the amount of sediment observed and the conversion rate of bidesmosidic and monodesmosidic was reduced by about 10% compared to two lots prepared by the standard process (Figures 2, 3, 4, 12, 14 - 16).
Based on the experimental results at laboratory (Examples 3 and 4) and pilot plant scale (Example 6), it can be concluded that the disclosed processes (boiling or bentonite treatment) allow to obtain an equilibrated relative amount of bidesmosidic and monodesmosidic saponins, mitigating the formation of sediments -unwanted in yucca extracts-. The same process could also be applied in other sources of steroidal glycosides, such as -but not limited to- Yucca brevifolia Engelm., Yucca baccata Torr., etc.
Example 7. Yucca monodesmosidic saponins as immunological adjuvant.
To determine the immunostimulant activity of the highly purified fraction containing monodesmosidic saponins obtained in the Example 5, female BALB/c mice were immunized with PBS buffer containing ovalbumin (OVA, 50 .g) (Control) and the following adjuvants (25 jig per dose): Freund's Complete Adjuvant (FCA), purified quillaja saponin QS-21 (>95% purity UHPLC-UV), and monodesmosidic yucca saponins (91.1% purity HPLC-ELSD). On weekly basis, four doses of antigen/adjuvant were applied to each group of mice (immunized intraperitoneally; seven mice per condition). Three days after the fourth immunization, blood samples were recovered from each mouse, and the titers of total IgG against ovalbumin (OVA) were determined by ELISA. As shown in Figure 11 , the relative titers of total IgG against OVA (expressed as titers relative to the control without adjuvant) induced by yucca monodesmosidic saponins was comparable at the same dose with the corresponding quillaja saponin QS-21 , a potent immunostimulant compound employed as adjuvant in vaccine formulations (p < 0.0001 ).
Claims
1 . A method to control the content of mono and bidesmosidic saponins in yucca extracts, suitable for isolation of steroidal saponins with immunostimulant properties, comprising: a) crushing and extruding fragmented harvested fresh or hydrated dried yucca biomass to obtain a first yucca juice extract (first precursor) having between 15 - 25 °Bx, such first yucca juice extract then is submitted to: a.1 ) heating up below the boiling point and a maximum temperature of 88 °C by steps to promote a controlled conversion and a precipitation of monodesmosidic saponins; or a.2) a one-step boiling at 100 °C or a heating above 100 °C, or a.3) a bentonite treatment, or a.4) performing sequentially step a.2) and step a.3) but cooling after step a.2) or vice versa, b) concentrating such first yucca juice extract obtained from step a) by either conventional evaporation or membrane concentration techniques or a combination thereof, to obtain a second yucca juice extract (second precursor) having 25 - 35 °Bx; c) filtering such second yucca juice extract after adjusted pH, preferably, with a pH buffer selected from H3PO4, HCI, citric acid, ascorbic acid or a mixture of one or more thereof, d) concentrating such filtered yucca juice extract, either by conventional evaporation, membrane concentration techniques or a combination thereof, to obtain a third yucca juice extract having 45 - 55 °Bx; and e) pasteurizing at 88 °C, such third yucca juice extract to obtain a fourth yucca juice extract having 45 - 55 °Bx and pH between 3.5 - 4.5, and then optionally, drying to be stored, wherein after step a.1 ), the method further comprising submitting such heated fresh or hydrated dried yucca juice extract to a controlled conversion at 40 - 30 °C.
2. The method of claim 1 wherein such fragmented harvested fresh or hydrated dried yucca biomass is selected from fragmented harvested fresh roots, stems, and leaves of yucca plant biomass or hydrated dried biomass.
3. The method of claim 1 or 2 wherein such yucca plant corresponding to genus Yucca comprising, including, but not limited to: Yucca schidigera Roezl., Yucca brevifolia Engelm., Yucca baccata Torr.
4. The method of claim 1 wherein step a.1) comprising heating up in 1 -10 steps.
5. The method of claim 4 wherein step a.1) comprising heating up in 2-3 steps.
6. The method of claim 1 wherein such first yucca juices (first precursor) having 19°Bx.
7. The method of claim 1 wherein such second yucca juice extract (second precursor) having 30 °Bx.
8. The method of claim 1 wherein such third yucca juice extract having 50 °Bx.
9. The method of claim 1 wherein such fourth yucca juice extract having 50 °Bx.
10. The method of claim 1 wherein such pH buffer is selected from H3PO4, HCI, citric acid, ascorbic acid, or a mixture the two or more thereof.
11 . The method of claim 1 wherein pH is 3.9.
12. The method of claim 1 wherein such temperature of controlled conversion is 35 °C.
13. The method of claim 1 wherein after step a.1 ) and either after steps b), c), or d), the method further comprises submitting the sediment obtained after filtering to a water washing and centrifugation step in 1 -5 cycles, and purified monodesmosidic saponins are separately recovered.
14. The method of claim 13 wherein such centrifugation step comprising 2-3 cycles.
15. The method of claim 1 wherein step a.3) comprising a treatment with a 0 - 100 g/L bentonite suspension.
16. A highly pure yucca monodesmosidic saponins precipitate having between 60-100% purity at HPLC-ELSD.
The highly pure yucca monodesmosidic saponins precipitate of claim 16 having between 70% - 100% purity at HPLC-ELSD. Use of a highly pure yucca monodesmosidic saponins precipitate having between 70% - 100% purity at HPLC-ELSD as immunological adjuvant.
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US2715122A (en) * | 1952-09-05 | 1955-08-09 | Edward S Rothman | Extraction of saponins from yucca baccata |
US4657766A (en) * | 1985-04-15 | 1987-04-14 | Distributors Processing Inc. | Yucca extract grain tempering mixture and process |
WO1995030009A2 (en) * | 1994-04-29 | 1995-11-09 | Gatsby Charitable Foundation | Identification, production and use of saponin glyclosyl hydrolases |
US20050106306A1 (en) * | 2003-09-12 | 2005-05-19 | Mennett Randall H. | Use of Labiatae herb preparations for foam enhancement of malt beverages |
US20070196517A1 (en) * | 2005-07-13 | 2007-08-23 | Dictuc S.A. | Modified Saponin Molluscicide |
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2022
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US2715122A (en) * | 1952-09-05 | 1955-08-09 | Edward S Rothman | Extraction of saponins from yucca baccata |
US4657766A (en) * | 1985-04-15 | 1987-04-14 | Distributors Processing Inc. | Yucca extract grain tempering mixture and process |
WO1995030009A2 (en) * | 1994-04-29 | 1995-11-09 | Gatsby Charitable Foundation | Identification, production and use of saponin glyclosyl hydrolases |
US20050106306A1 (en) * | 2003-09-12 | 2005-05-19 | Mennett Randall H. | Use of Labiatae herb preparations for foam enhancement of malt beverages |
US20070196517A1 (en) * | 2005-07-13 | 2007-08-23 | Dictuc S.A. | Modified Saponin Molluscicide |
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