# US 8,568,798 B2 · Production and extraction of procyanidins from plant cell cultures

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- Number: US 8,568,798 B2
- Document ID: US8568798B2
- Status: Granted
- Kind: B2
- Application number: 13/262,456
- Filed: 2009-05-04
- Published: 2012-01-26
- Granted: 2013-10-29
- Earliest claimed: 2009-04-03
- Assignees: Dianaplantsciences, Inc.
- Applicants: Mylavarapu Venkatramesh; D. Ry Wagner; Sonia Lall; Frederic Y. Lejard; Sung-Yong H. Yoon
- Inventors: Mylavarapu Venkatramesh; D. Ry Wagner; Sonia Lall; Frederic Y. Lejard; Sung-Yong H. Yoon
- Web page: https://patentgrep.com/patent/US8568798B2

## CPC

- A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 39/06: Free radical scavengers or antioxidants
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided herein are methods of making cocoa polyphenol preparations, which methods comprises harvesting cocoa polyphenols, for example, procyanidins, from the cell suspension culture. In examples of these methods, the resultant cocoa polyphenol preparation is substantially (or in some cases, completely) free of detectable caffeine and theobromine, and more generally substantially free of xanthine alkaloids. Methods of producing a cell suspension culture of cacao cells are also described, including cell suspension cultures useful for making cocoa polyphenol and, more specifically, procyanidin preparations. Theobroma and Herrania sp cell suspension cultures and cocoa polyphenol preparations made therefrom are also provided, in particular xanthine alkaloid-free (or caffeine- and/or theobromine-free) cocoa polyphenol preparations.

## Claims

### Claim 1

A method of producing a cell suspension culture of cacao cells, comprising:
growing callus from a Theobroma cacao plant on growth medium to form a callus culture;
identifying and selecting a rapidly growing cell line from the cells of the Theobroma cacao callus culture, the rapidly growing cell line selected to produce procyanidins in cell suspension with less than 2% theobromine and/or less than 0.5% caffeine contamination on a dry weight basis; and
initiating the cell suspension culture by inoculating the rapidly growing cell line into liquid medium and producing in cell suspension procyanidins with less than 2% theobromine and/or less than 0.5% caffeine contamination on a dry weight basis.

### Claim 2 (depends on 1)

The method of claim 1, wherein the Theobroma cacao plant is a floral explant selected from the group staminode, sepal and petal base explants.

### Claim 3 (depends on 1)

The method of claim 1, further comprising growing the cell suspension culture in flasks, any suitable culture vessels, or bioreactors.

### Claim 4 (depends on 3)

The method of claim 3, wherein the growing is effected in vessels or bioreactors and in batch, fed-batch or continuous mode.

### Claim 5 (depends on 1)

A Theobroma cacao cell suspension culture produced by the method of claim 1.

### Claim 6 (depends on 1)

The method of claim 1, wherein the growth medium is the liquid media XXXIII listed in Table 1.

### Claim 7 (depends on 1)

The method of claim 1, wherein the callus is from a vegetative material.

### Claim 8 (depends on 7)

The method of claim 7, wherein the vegetative material is selected from leaves, stems, meristem, nodes, or internodes that are young or mature.

### Claim 9 (depends on 1)

The method of claim 1, wherein the step of selecting the rapidly growing cell line includes analyzing the callus culture using high performance liquid chromatography to determine the production of procyanidins.