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United States Patent (10) Patent No.: US 7,108,870 B2 Sangwan Et Al

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United States Patent (10) Patent No.: US 7,108,870 B2 Sangwan Et Al US00710887OB2 (12) United States Patent (10) Patent No.: US 7,108,870 B2 Sangwan et al. (45) Date of Patent: Sep. 19, 2006 (54) PROCESS FOR ISOLATION OF 2004/0033273 A1 2/2004 Patwardhan et al. ........ 424,725 WITHAFERN-AFROM PLANT MATERALS 2004/0096524 A1* 5, 2004 Nair et al. ... 424,725 AND PRODUCTS THEREFROM 2004/0258781 A1* 12/2004 Nair et al. .................. 424,769 (75) Inventors: Rajender Singh Sangwan, Lucknow FOREIGN PATENT DOCUMENTS (IN): Narayan Das Chaurasiya, JP 2002O187846 * 7/2002 Lucknow (IN); Laxmi Narayan Misra, Lucknow (IN); Payare Lal, Lucknow OTHER PUBLICATIONS (IN). Girish Chandra Uniyal, Dinan et al. J. Chromatogr. A. 2001. vol. 935, pp. 105-123.* Lucknow (IN); Neelam Singh Kaufmann et al. Chromatographia. 2001. vol. 54, pp. 394-398.* Sangwan, Lucknow (IN); Avdhesh Kumar Srivastava, Lucknow (IN); * cited by examiner Krishan Avtar Suri, Jammu (IN); Primary Examiner Christopher R. Tate Ghulam Nabi Qazi, Jammu (IN); (74) Attorney, Agent, or Firm—Buchanan Ingersoll & Rakesh Tuli, Lucknow (IN) Rooney, PC (73) Assignee: Council of Scientific & Industrial (57) ABSTRACT Research, New Delhi (IN) (*) Notice: Subiect to any disclaimer, the term of this The present invention relates to an improved process of t i f ded d ted under 35 analytical and quantitative isolation of withaferin-A from pp l s 95. justed under Withania Somnifera (Sanskrit: Ashwagandha, English: win M YW- (b) by yS. ter cherry) and other plants and products therefrom, said (21) Appl. No.: 10/822,858 method comprising steps of selecting most appropriate extraction medium composition, with extraction solvent (22) Filed: Apr. 13, 2004 system consisting of a defined admixture of water and alcohol in a range of proportion of alcohol within a narrow (65) Prior Publication Data range of (methanol, ethanol etc.) (preferably 60% alcohol and 40% water) for the dry plant material and from 60% to US 2005/022695O A1 Oct. 13, 2005 80% alcohol in the alcohol-water mixture for fresh plant material, the invented extractant composition leading to (51) Int. Cl. better/improved yields and accurate quantitative estimations A6 IK 36/00 (2006.01) of withaferin A existent in planta or in materia, selecting A6 IK3I/35 (2006.01) effective state of the tissue particularly use of the fresh (52) U.S. Cl. ....................................... 424/725; 514/453 materials improving yields, providing logistic benefits of (58) Field of Classification Search ................ ... None isolations and diagnosis, and according several other advan See application file for complete search history. tages including technological, economic, preventing con (56) References Cited taminations etc., the said method being valuable and advan tageous for standardization of botanicals, metabolomic U.S. PATENT DOCUMENTS characterization of plants particularly chemotypes, analyZ ing metabolic transitions under physiological, genetical, 5,494.668 A * 2/1996 Patwardhan ................ 424,756 environmental and biotic perturbations. 6,153,198 A * 1 1/2000 Ghosal ....................... 424,773 6,713,092 B1 3/2004 Ghosal ....................... 424,725 6,866,872 B1* 3/2005 Tomi et al. ................. 424,725 14 Claims, 1 Drawing Sheet U.S. Patent Sep.19, 2006 US 7,108,870 B2 s * & & ). s 1 2 3 4 5 67 8 9 10 FIG US 7,108,870 B2 1. 2 PROCESS FOR ISOLATION OF specific lable claims but also assert Stringencies of compli WITHAFERN-AFROM PLANT MATERALS ances of regulatory frameworks like compliances like AND PRODUCTS THEREFROM DSHEA, NHPR and other analogous regulatory acts and statutes. To cater to these and other needs, Therefore, FIELD OF THE INVENTION appropriateness of the methodolocal processes for extrac tions described herein is not only of tremendous importance The present invention relates to an improved process for for the better and improved yields, resource characterization analytical and quantitative isolation of withaferin-A from and accurate quantitative estimations existent in planta or in plant materials and products therefrom, said method com material, but also provides benefits and logistics of isola prising steps of selecting effective extractant solvent system, 10 tions, diagnosis, metabolomics etc. The process is valuable with extraction solvent system consisting of a defined and advantageous in global scale of applications and rel admixture of water and alcohol in a range of proportion of evance for standardization of ashwgandha botanicals, alcohol (methanol, ethanol or other alcohol) with compa metabolomic characterization of plants, chemotypes, ana rable nature of polarity, dielectic constant and dipole lyzing the nature and pace of metabolic transitions under moment etc.) from 60 to 70% (preferably 60%) for dry 15 physiological, genetical, environmental and biotic perturba material and from 60 to 80% (preferably 60%) for fresh tions etc. material, with the fresh material process providing improved Withanolides or withasteroids are a group of naturally recovery and more accurate estimations, process with aque occurring Cs-steroidal lactone molecules of basically trit ous extraction of withaferin-A and augmentation of recovery erpene ancestory and are structurally based on an intact or of withaferin-A from Such aqueous extraction residues also rearranged ergostane framework. The characteristic lactone forms the part of the invented process, with processes and is formed through conjoining of Ca hydroxyl and C. methods involving different extractant system leading for carboxylic acid function. Thus, chemically withanolides are ways and means of improvement of withaferin-A recovery defined as 22-hydroxy ergostane-26-oic acid 26.22-lactone. and its accurate estimation etc. from different states of Biogenesis of withasteroids is known to occur highly biomass and products therefrom, better yields and quantita 25 scarcely like Tacca plantaginea of Taccaceae (Z. L. Chen, B. tive estimations of withaferin-A existent in planta or in D. Wang and J. H. Shen, 1988, Phytochemistry 29, 2999), material in conjunction with state of the tissue specific Cassia siamae of Leguminoseae (C. Srivastava, I. R. Sid extraction systems including use of the fresh materials for diqui, J. Singh and H. P. Tewari, 1992, J. Ind. ChemSoc. 69, improving yields/estimations providing logistic benefits of 111), Ajuga parviflora of Labiatae (P. M. Khan, S. Ahmad, isolations and diagnosis, and according several other advan 30 H. Rubnawaz and A. Malik, 1999, Phytochemistry, 51, 669; tages including technological, economic, preventing con P. M. Khan, S. Ahmad and H. R. Nawaz, 1999, J. Nat. Prod. taminations etc., the said method being valuable and advan 62, 1290; H. R. Nawaz, A. Malik, P. M. Khan and S. Ahmad, tageous for, metabolomic characterization of plants 1999, Phytochemistry, 52, 1357) beyond their usual species particularly chemotypes, analyzing metabolic transitions limited prodigal producers i.e. 16 out of 96 genera of potato under physiological, genetical, environmental and biotic 35 family (Solanaceae). These include, besides the well known perturbations in the plant and materials of Withania and Ayurvedic, Sidha, and Unani medicinal plant, Ashwagandha other congener species and for standardization of botanicals, (winter cherry, Withania somnifera), Lycium, Datura, products and formulations therefrom for better label claims Iochroma, Physalis, Tubocapsicum, Solanum etc. Most of and accurate rating. Thus, the essence of field of invention these plants are valued and/or used as: herbs or health foods is development of a process for efficient isolation and 40 and/or medicinal plants (based on the traditional knowledge accurate quantitative estimation of withaferin-A (a withano of their positive Support to body functions and responses), lide or withasteroid) in cells, plants, tissues and products, healing and healthful materials (as such or in the form of preparations and formulations therefrom using metabolomic Supplements, nutraceuticals, concoctions, formulations and techniques comprising precise extraction conditions and compositions) althrough individuals age of growing and state of the tissues, liquid-liquid partitioning, flash-evapo 45 graying, rejuvenating and as remedial herbal products of ration, sample preparation and analytical high performance acceptance, Source of valuable phytoceuticals/phytochemi liquid chromatographic resolution and quantification in said cals with potentials of lead multifunction drugs/nutraceuti method to determine quantitative, qualitative, native com cals of near future. Withaferin-A (ergostanically named as position of materials and products therefrom. 4f3, 27-dihydroxy-5?, 6(3-epoxy-1-oxowitha-2.24-dieno 50 lide), the first member of this group of compounds was BACKGROUND AND PRIOR ART REFERENCE isolated from the well known Indian medicinal plant Ash wagandha (P. A. Kurup, 1956, Antibiotic principle of the Ashwagandha is one of the most valuable medicinal leaves of Withania somnifera, Current Science, India, 25: plants, forming the active component or whole of the 57) and its structure elucidated in 1965 (D.Lavie, E. Glotter phytopharmaceutical products for the prevention and cure of 55 and Y. Shvo, 1965, Constituents of Withania Sonnifera a variety of human ailments and diseases. Numerically, Dun the structure of withaferin A. J. Chem. Soc. 7517). Ayurvedic, Unani and Sidha medicinal products based on Besides it is known to occur as one of the major withano the plant currently run above one hundred and it is rapidly lides in other Withania species (W. aristica,
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