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(12) Patent Application Publication (10) Pub. No.: US 2005/0196409 A1 Da0 Et Al

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US 2005O1964O9A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0196409 A1 Da0 et al. (43) Pub. Date: Sep. 8, 2005 (54) COMPOSITIONS OF BOTANICAL (52) U.S. Cl. ....................... 424/195.15; 435/6; 435/723; EXTRACTS FOR TREATING 424/741; 424/746; 424/769; MALIGNANCYASSOCIATED CHANGES 702/20 (76) Inventors: James Dao, Henderson, NV (US); Jeffrey J. Dao, San Mateo, CA (US) Correspondence Address: (57) ABSTRACT MORRISON & FOERSTER LLP 755 PAGE MILL RD PALO ALTO, CA 94304-1018 (US) Methods for diagnosis of malignancy associated changes (MAC) using Automated Quantitative Cytometry (AQC) (21) Appl. No.: 10/949,178 and treatment using compositions, extracts and compounds comprising botanical extracts. Use of Such compounds in the (22) Filed: Sep. 24, 2004 prevention and therapy of cancer diagnosed by the AOC Related U.S. Application Data MAC test are also provided as well as methods for treatment using the compositions of this invention. Compositions (60) Provisional application No. 60/506,066, filed on Sep. comprising therapeutically effective amounts of two or more 24, 2003. of an extract of Ganoderma lucidum, an extract of Salvia miltiorrhiza and an extract of Scutellaria barbata and Publication Classification optionally a therapeutically effective amount of an extract of Hippophae rhamnoides are provided. Novel Synergistic (51) Int. Cl." ......................... A61K 35/84; A61K 35/78; effects of the use of these compounds in combination C12O 1/68; G01N 33/574; therapy are disclosed. Compositions further comprising G06F 19/00; G01N 33/48; therapeutically effective amounts of at least one chemothera GO1N 33/50 peutic agent are also provided. Patent Application Publication Sep. 8, 2005 Sheet 1 of 31 US 2005/0196409 A1 NOLOWLXCCLT? W. RIIV?NLARICI NOILOVILXIILVIAWHO CINQORIÐ8IRIGHTH/{TRIGIHQINGHT|8H NOILOVRILXGI' (HJLWRIALTIHsarios NOILVRILNGIONOO/NOILnTIGI CHOWRIOLSSISÄTVNVGITAWVS(HOH SOITONGIH?SLNVQIXOILNV FIGURE 1A Patent Application Publication Sep. 8, 2005 Sheet 2 of 31 US 2005/0196409 A1 FIGURE 1B westeresfroi SB Tree 1. Cut leave with scissors 2. or pull of directly Purchased hers: Store at -20C FREEZEORYTNG MLLNG Freeze to -50°C then Wiley Mill, Screen a lmm Dry at Shef Temps 20-22°C Blade Spacings 0.35mm Condenser s 50°C, Vac's 2100Hg Speed = 1250 rpm Store solid at -20°C for subsequent analysis EXTRACON Ground herb (5g)+boiling HO FREEAEORYNG (150 ml) (18 Mohm), Stir magnetically, Freeze to SOC 35 min at reflux in 250 ml RBF with condenser Dry at: Shef Tenp = 20-22°C Condensers -50°C, Vacs 100pHg FILTRATION (250nal Suction Flask) 9c. Bichner Fine CENTRFUGATON 3X9 cm Kendall AG milkfilters, layered Transfer contents to bottle (Sorval Dry-Spi Filter boiling mixture Centrifuge Bottle) with 15ml HO Rinse 250 m flask with 25ml boiling HO Centrifuge (10,000 rpm, 20 min, 28-32°C) Rinse filter cake with 25ml boiling HO Decantation Air Dry residue in bottle Air Dry filter Cake Decant Supernatant and bringwolume to 250ml with H2O Sample immediately for Antioxidant Analysis, Store east 20°C Conubine Solids and Store at -20C Patent Application Publication Sep. 8, 2005 Sheet 3 of 31 US 2005/0196409 A1 FIGURE 1C EXTRACTION Ground herb (5 g) + EtOHIHO (80%, whv), 125 ml) Stirlh in 250 ml. RBF FILTRATION (250 ml Suction Flask) 5%.cm Bochner Funnel Whatman GFA atop # 1 filter paper Filter mixture Rinse flask with 25n 80% EtOH Sample for Antioxidant Analysis Rise filter cake with 25ml 80% EtOH Store remainder at -20°C A DLUTION Filtrate Dilute to 250 ml with 80% EtOH Filter cake CONCENTRATION RE-EXTRACTION Rotary evaporation Or B Extract filter cake with 35C, Vacuum 228"Hg 2S 80%EOH for Leave residue under vacuun Dry to constant weight Store residine at -20°C for Subsequeat analysis Filter cake STORAGE -20°C Patent Application Publication Sep. 8, 2005 Sheet 4 of 31 US 2005/0196409 A1 FIGURE 1D KTRACTION Ground herb (10 g) + CHC/MeOH (50%, viv), 50 mi Stir and reflux 35 min in 250 ml. RBF FLTRATION (250 ml. Suction Flask) 5Actin Bochner Funnel Whatman GFA atop fit filter paper Filter mixture Rinseflask with 25ml 50% CHC/MeOH Rinse filter cake with 25ml 50% CHCMeOH CONCENTRATION Rotary evaporation 30°C, Vacuum 228"Hg Filtrate Filter cake DLUTON RE-EXTRACTION Add 100 m. CHC+ Extract filter cake with 100 m do to concentrate 150 ml 50%CHC/MeOH for 35 min SEPARATION LTRATON Transfer to sep. funnel (500 ml), As above Rinse flask X50 midd-O, 2X50 ml CHC. IX50 ml ddHO, Shake mixture and separate layers Filtrate Filter cake Bottom Organic Layer CONCENTRATION FREEZEORYNG Rotary evaporation Freeze to -50C 25°C, Vacuum-28"Hg Dry at Shelf Temp = 20-22°C Condenser = -50°C, Wac = . >00Hg Patent Application Publication Sep. 8, 2005 Sheet 5 of 31 US 2005/0196409 A1 FIGURE 1E EXTRACTION Ground SB Berry (1.67 g) + Ground SB Leaf (1.67g)+Powdered NA Ginseng Extract (1.68g) + Boiling HO (150 ml), (18Mohm)). Stir magnetically 35 min at reflux in 250 ml RBF with Condenser FTLTRATION (250 ml Suction Flask) 9can Bochner Funnel 3X9 crin Kendall AG milk filters, layered Filter boiling mixture Rinse 250 ml flask with 25ml boiling HO Rinse filter cake with 25ml boiling Ho Filter Cake CENTRFUGATION Transfer contents to bottle (Sorvall Dry-Spin' 2 EXTRACTION Centrifuge Bottle) with 15ml HO 3 Filtrate Extract wet filter cake with Centrifuge (10,000 rpm, 20 min, 28-32°C) 150 ml boiling HO (18 Mohm) for 35 min as above SUPERNATANTS (1,2,3) Air Dry residues in bottle Decant supernatant and bring volume to 250 ml with HO 3'EXTRACTION Sample for immediate antioxidant analysis or Extract wet filter cake with store ut-20°C for future analysis 150 ml boiling HO (18 Mohm) for 35 mini and filter as above 3 Filtrate Patent Application Publication Sep. 8, 2005 Sheet 6 of 31 US 2005/0196409 A1 FIGURE 1F EXTRACTION Ground SB Berry (1.67 g) + Ground SB Leaf (1.67g)+Powdered NA Ginseng Extract (1.68g) + BeOH/HO(80%, wiv), 150 ml) Stir magnetically lhat reflux in 250 ml. RBF with Condenser FILTRATION (250 ml. Suction Flask) 9cm Beichner Funnel 3X9 cm Kendall AG milk filters, layered Filter boiling mixture Rinse 250 m flask with 25m1.80%EtOH Rinse filter cake with 25m 80%EOH Filter Cake OLUTION ad Transfer filtrate and dilute to 2 EXTRACTION 250 m with 80% EtOH Extract wet filter cake with 150 ml 80% BOH for has above Sample for immediate antioxidantanalysis or store at -20C for future analysis Extract wet filter cake with 150 ml 80% BOH for has above 3"Filtrate Patent Application Publication Sep. 8, 2005 Sheet 7 of 31 US 2005/0196409 A1 FIGURE 1G 1 EXTRACTION Ground SB Berry (1.67 g) + Ground SB Leaf (1.67g)+Powdered NA Ginseng Extract (1.68g) + Boiling HO (150 ml), (18Mohm) Stir magnetically 35 min at reflux in 250 ml RBF with Condenser FELTRATION (250 ml Suction Flask) 9can Bochner Funnel 3X9 cm Kendali AG milk filters, layered Filter boiling mixture Rinse 250 ml flask with 25rial boiling HO Rinse filter cake with 25ml boiling HO Filter Cake CENTRFUGATION r 2 EXTRACTION Transfer contents to bottle (Sorvall Dry-Spin' Centrifuge Bottle) with 15ml HO Extract wet filter cake with Centrifuge (10,000 rpm, 20 min, 28-32°C) 150 ml EtOH/HO(80%, viv) for has above SUPERNATANT Decant supernatant and bring FILTRATION (250 ml Suction Flask) wolume to 250 with Scanlochner Fune HO Whatman GFA atop fl filter paper Filter mixture Rise fask with 25m18.0%BOH Rise filter cake with 25m 80%EtOH Sample for immediate antioxidantanalysis or store at -20°C for future analysis Patent Application Publication Sep. 8, 2005 Sheet 8 of 31 US 2005/0196409 A1 Raw botanical material inspected and sorted for uniform size and quality. Tested for lack of heavt metals. Raw botanical material extracted with ethyl acetate (solvent). o solvent to raw material ratio between 5:1 and 9:1 e extraction in agitating vessel (stainless steel or lined with glass) at 50-100 rpm, at 50-75 °C, under 1-1.2 ATM pressure. The procedure is repeated once under identical conditions. Extracts from both extraction steps are collected and filtered to ensure removal of solids. Liquid extracts are reduced by evaporation to about 5% of original volume O solvent material is removed Concentrated extracts are evaporated to dryness under vacuum using extruded evaporator or by spray drying O solvent material is removed Recovered solid products are blended and optionally, encapsulated FIGURE 1H Patent Application Publication Sep. 8, 2005 Sheet 9 of 31 US 2005/0196409 A1 uopeu||quoosexepu?(10)senjeaJo?uop?q??ul?o lleouopela?||oud(GHS)sise?uosuopeu||quoojosioenx= +y)+6) pue(s) uue?sæM sônudpex?u Je J?au}OGOI senjea Patent Application Publication Sep. 8, 2005 Sheet 10 of 31 US 2005/0196409 A1 :weaunela quo ve (fu?u) one A Ogof Patent Application Publication Sep. 8, 2005 Sheet 11 of 31 US 2005/0196409 A1 ÉÐ%9eHo?ayozHul.szyo nozHojawooviziulugv?žºš?m| Patent Application Publication Sep. 8, 2005 Sheet 12 of 31 US 2005/0196409 A1 u?u001,1o HOÐUN/ZTOZHO,Gw u?uZ/Off<O OZH/OVO?E/MAL Patent Application Publication Sep. 8, 2005 Sheet 13 of 31 US 2005/0196409 A1 C O C O O O O C C er O C C en vu . er en . vur (auru) (u?u) saneA sents A OSO/I paenots). OSO/ potents) Patent Application Publication Sep. 8 2005 Sheet 14 of 31 US 2005/0196409 A1 soneA os Of paeneo Patent Application Publication Sep. 8, 2005 Sheet 15 of 31 US 2005/0196409 A1 O f O A G en y t I XOXOO Patent Application Publication Sep.
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