US007815949B2 (12) United States Patent (10) Patent No.: US 7,815,949 B2 Cohen (45) Date of Patent: Oct. 19, 2010 (54) ESTROGENIC EXTRACTS OF MORUS ALBA 6,348,204 B1 2/2002 Touzan AND USES THEREOF 6,551,627 B1 4/2003 Yoon et al. FOREIGN PATENT DOCUMENTS (75) Inventor: Isaac Cohen, Piedmont, CA (US) CN 1105252 A 7, 1995 (73) Assignee: BioNovo, Inc., Emeryville, CA (US) N E. A R (*) Notice: Subject to any disclaimer, the term of this N o g 3. patent is extended or adjusted under 35 JP 2001.002568 1, 2001 U.S.C. 154(b) by 473 days. JP 2002142717. A 5/2002 JP 2004 189609 A T 2004 (21) Appl. No.: 11/298,080 WO WO 2004/OO6946 * 1, 2004 (22) Filed: Dec. 9, 2005 OTHER PUBLICATIONS O O Supplemental EP Search Report mailed Apr. 14, 2008. (65) Prior Publication Data Ray et al., “Histological study on the effect of phytoestrogen on uterus and ovary of animal.” 196601.01, Indian Science Congress US 2006/0134245A1 Jun. 22, 2006 Association Proceedings 53(nr3);461, ISSN 0085-1817. Related U.S. Application Data EP 05853.308.4 Official Action mailed Jul. 13, 2010. AV * cited by examiner (60) Provisional application No. 60/637,302, filed on Dec. y 17, 2004. Primary Examiner Christopher R Tate Assistant Examiner—Randall Winston (51) Int. Cl. (74) Attorney, Agent, or Firm Wilson Sonsini Goodrich & A6 IK36/00 (2006.01) Rosati A6 IK9/00 (2006.01) A6 IK 47/00 (2006.01) (57) ABSTRACT (52) U.S. Cl. ....................... 424/774; YSE Extracts of various species of the Moraceae family have estro 58) Field of Classification S h s N genic properties. For example, aqueous and ethanolic extracts (58) O li assi sate sts - - - - - - - - - -hhi - - - - - - - O of Morus alba L. Species possess estrogenic properties in both ee application file for complete search history. ERC+ and ERB+ cells. These estrogenic effect include estro (56) References Cited gen response element (ERE) stimulation as well as tumor necrosis factor (TNF) repression. Methods are provided for U.S. PATENT DOCUMENTS treating climacteric symptoms, breast and/or uterine cancer, 5,164,182 A * 1 1/1992 Meybecket al. ............ 424,773 and osteoporosis. 5,674,497 A * 10/1997 Kuwana et al. ............. 424,765 6,280,715 B1 8/2001 Seguin et al. 5 Claims, 1 Drawing Sheet U.S. Patent Oct. 19, 2010 US 7,815,949 B2 FIG. 1 U937 Ces 100 Luciferase ERC lug Activity A ER? lug 75 • ERa + ERB Fold of Control SO 25 0 10 - 10 - 10 10 10 10 Estradiol, (M) FIG. 2 MDA-MB-435 Cells 40 ERa lug Luciferase A ERB lug Activity 30 ERa + ERB Fold of Control 20 10 O O 10 10 - 10 10° 10 10 Estradiol), (M) US 7,815,949 B2 1. 2 ESTROGENC EXTRACTS OF MORUS ALBA tion failed. White mulberry is now grown in the United States AND USES THEREOF as an ornamental, as a windbreak for edging fields, and for its fruit (although the fruit of black mulberry is preferred over CROSS-REFERENCE that of white mulberry). In some parts of the southern United States, it is considered an invasive plant. It is readily adapted This application claims the benefit of U.S. Provisional to growing in a number of soil types, elevations and latitudes. Application No. 60/637,302, filed Dec. 17, 2004, which is There is no known report of using extracts of Morus alba L. incorporated herein by reference in its entirety. as estrogenic compositions. There is a need forestrogenic compositions that are readily FIELD OF THE INVENTION 10 obtained from natural sources. There is also a need for meth ods of making Such compositions. There is also a need for The present invention relates to plant extract compositions, methods of using Such estrogenic compositions. and more particularly to compositions comprising extracts of plant species belonging to the species Morus alba L. (white SUMMARY OF THE INVENTION mulberry). The invention further relates to methods of using 15 and methods of making Such plant extract compositions. The invention provides a plant extract composition that contains an extract of a plant species of the species Morus BACKGROUND OF THE INVENTION alba L. The invention also provides a method of eliciting an estro Hormone replacement therapy (HRT) has been used suc genic effect in a Subject. The method includes administering cessfully to treat a variety of conditions, such as osteoporosis, to a Subject an estrogenically effective amount of the estro increased risk of cardiovascular disease in post-menopausal genic Morus alba L. extract composition. women and climacteric symptoms, such as hot flashes, The invention further provides a method of activating decreased libido and depression. However, HRT with estra estrogen response element (ERE). The method includes con diol (E), either alone or in combination with progestin, can 25 tacting a cell, which has both a gene under control of an lead to undesirable effects. In fact, a recent Women's Health estrogen response element and an estrogen receptor, with an Initiative (WHI) study was abruptly halted when preliminary amount of the inventive Morus alba L. extract composition results showed that HRT was associated with a 35% increased that is effective to activate the gene through interaction of the risk of breast cancer. ER with the estrogen response element. Breast cancer can be treated or prevented by using a so 30 The invention further provides a method of repressing a called selective estrogen receptor modulator (SERM), such as gene under control of a tumor necrosis factor response ele tamoxifen. (Before the approval of tamoxifen, breast cancer ment (TNF-RE). The method includes administering to a cell, treatment of pre-menopausal women often included remov which has a TNF response element (TNF-RE) operatively ing the ovaries in order to reduce the cancer-stimulating effect linked to a gene, an amount of the inventive Morus alba L. of estrogen.) Tamoxifen appears to selectively block the can 35 extract composition that is effective to repress expression of cer-inducing effects of estrogen inbreast tissues of pre-meno tumor necrosis factor. In some embodiments, the gene is pausal women. Another SERM, raloxifene, has been TNF-C. In other embodiments, the gene is a reporter gene. approved for treatment of osteoporosis as an alternative to The invention further provides a method of making the estrogen replacement. In addition to selectively inducing inventive Morus alba L. extract composition. The method estrogenic effects in bone tissue, long-term administration of 40 begins with obtaining plant matter from a plant of the species raloxifene was also shown to be associated with reduction in Morus alba L. The method continues with contacting the the rate of breast cancer in the Multiple Outcomes of Ralox plant matter from a plant species of the species Morus alba L. ifene Evaluation (MORE) study. with an extraction medium under conditions Suitable to form While SERMs such as tamoxifen and raloxifene provide an extract solution. The method then provides for separating selective reduction in estrogen's cancer-inducing effects in 45 the extract solution from the plant matter, and optionally the breast, they are not without their risks. For example both reducing or diluting the extract solution, thereby forming the tamoxifen and raloxifene therapy have been associated with extract. When reduced, the extraction solution can be either a increased incidence of hot flushes; and tamoxifen therapy has concentrate or a solid residue (residue). Whether reduced or been shown to increase the risk of uterine (endometrial) can not, the extraction solution, concentrate and residue are C. 50 referred to collectively as an “extract'. Despite the Success of estrogen replacement therapy in treating osteoporosis, coronary heart disease and climacteric INCORPORATION BY REFERENCE symptoms, and despite the Success of using SERMs like All publications and patent applications mentioned in this tamoxifen and raloxifene in treating breast cancer and specification are herein incorporated by reference to the same osteoporosis, there remains a need for compositions having 55 extent as if each individual publication or patent application estrogenic properties. Additionally, given the increasing cost was specifically and individually indicated to be incorporated of producing drug compounds, there is a need for additional estrogenic compositions that may be obtained from natural by reference. SOUCS. BRIEF DESCRIPTION OF THE DRAWINGS Various cultivars of white mulberry (Morus alba L.) have 60 been grown throughout the world for a wide variety of pur The novel features of the invention are set forth with par poses. It generally grows as a tree or a shrub. In the Orient, it ticularity in the appended claims. A better understanding of has been grown for centuries for its leaves, which are used as the features and advantages of the present invention will be silk worm fodder, and secondarily as fodder for herbivorous obtained by reference to the following detailed description, livestock. In the United States, cultivation is believed to have 65 which sets forth illustrative embodiments, in which the prin been first attempted by Shakers, who intended to use the ciples of the invention are utilized, and the accompanying leaves as fodder for silk worms, however silkworm cultiva drawings of which: US 7,815,949 B2 3 4 FIG. 1 is a graph of luciferase expression in U937 (human antibody that only detects ERC. Thus, most studies examin monocyte) cells transformed with DNA encoding estrogen ing the effects ERS in breast tumors on clinical outcomes response element linked to the minimal thymidine kinase (tk) reflect the only ERC. status. However, several recent studies promoter and a sequence encoding luciferase (Luc) in have detected the presence of ERB mRNA in human breast response to varying concentrations of estradiol (E) in the 5 tumors.