WO2013137822A1

WO2013137822A1 - Methods and systems for detoxifying phorbol esters in plant products

Info

Publication number: WO2013137822A1
Application number: PCT/SG2013/000084
Authority: WO
Inventors: Yan Hong; Yunping BU; Loong Chueng LO
Original assignee: Temasek Life Sciences Laboratory Limited
Priority date: 2012-03-12
Filing date: 2013-03-01
Publication date: 2013-09-19
Also published as: AP2016009027A0

Abstract

[0083] The present invention relates to methods and systems for detoxifying phorbol. esters in plant products or materials, particularly in plant products or materials from the Euphorbiacaeae family or the Thymelaeaceae family, particularly in the genera Jatropha, Croton, Hippomane, Wikstroemia, Daphne or Pimelea, and more particularly in plant products or materials from Jatropha curcas, Croton tiglium, Hippomane mancinella, Wikstroemia canescens, Daphne mezereum or Pimelea prostrate.

Description

METHODS AND SYSTEMS FOR

DETOXIFYING PHORBOL ESTERS IN PLANT PRODUCTS

BACKGROUND OF THE INVENTION

[0001] The present invention relates to methods and systems for detoxifying phorbol esters in plant products or materials, particularly in plant products or materials from the Euphorbiacaeae family or the Thymelaeaceae family, particularly in the genera Jatropha, Croton, Hippomane, Wikstroemia, Daphne or Pimelea, and more particularly in plant products or materials from Jatropha curcas, Croton tiglium, Hippomane mancinella, Wikstroemia canescens, Daphne mezerewn or Pimelea prostrate.

[0002] The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.

[0003] Jatropha curcas (Jatropha) is a member of Euphorbiacaeae family. It is a shrub that has been used in tropical and subtropical regions of Central America, Asia and Africa as "living fences" around fields and settlements or folk medicine. It is gaining popularity as a biofuel feedstock plant due to its non-edible seed oil, drought tolerance and adaptability to almost any type of soil, including those in arid and marginal land that are not suitable for crops. It was projected that as much as 25.6 million tons of Jatropha oil could be produced yearly by 2015 (GEXSI 2008). Production of Jatropha oil (-30% of seed) also produces at least twice the amount of seed cake. Having a high protein content and amino acid profile similar to that of soybean meal, Jatropha cake holds great potential as a source of protein and other nutrients for livestock (Devappa et al., 2010a). Seed cake for livestock feeding will also increase commercial value of Jatropha plantation. However, antinutrients like trypsin inhibitor, lectin and phytate (Martinez-Herrera et al., 2006), and two major toxic components - curcin and phorbol esters (PEs), are present in Jatropha seeds (Makkar and Becker, 1997).

[0004] Curcin belongs to the ribosome inactivating proteins (RJPs) group. It shows cell- free protein synthesis inhibition, but not under in vivo conditions. Also, it is heat labile and easily degraded in soil (Gubitz et al., 1999; Lin et al., 2003; Devappa et al., 2010b). On the other hand, PEs are tetracyclic tiglian diterpenoids that are naturally produced by plants of the Euphorbiacaeae family (examples such as Jatropha curcas, Croton tiglium, Hippomane mancinella) and Thymelaeaceae {Wikstroemia canescens, Daphne mezerewn, Pimelea prostratd) family. Six different PEs have been characterized from Jatropha (Haas et al., 2002; Devappa et al., 2010b). Unlike curcin that is water soluble, PEs are hydrophobic (insoluble in water) molecules and present in both oil and seedcake (up to 8 mg/g in oil and 3 mg g in defatted kernel meal) and are heat stable. Since PEs are epidermal cell irritating and cancer promoting, Jatropha seed cake cannot be used directly as an animal feed without detoxification. Currently, they are often used as an organic fertilizer and increase of Jatropha seed yield between 13 to 120% was reported after returning Jatropha seedcake to Jatropha plantation. Not only do PEs reduce commercial values of Jatropha seed meal, they also cause some safety and environment concerns. PE containing Jatropha oil are hazardous to workers and there is also the concern of safety of crude jatropha oil (CJO) derived biodiesel if PE remains. As PEs are heat stable, there are also concerns on their possible ecotoxicity to other organisms when seed meals are used as fertilizers, or when seeds remain in soil for a prolonged period of time, with possible PE leaching into soil and being carried to water bodies.

[0005] Detoxification of jatropha oil and press-cake has been demonstrated at the laboratory scale (Haas and Mittelbach, 2000; Devappa and Swamylingappa, 2008; Kumar et al., 2008). The resulting detoxified seed meal was fed to fish and small rodents which showed only slightly lower growth rate than with standard rations when non-phorbol ester containing jatropha seed meal was used. (Gross et al., 1997; Devappa and Swamylingappa, 2008). Recently, a new process for removing detoxifying phorbol esters in jatropha seed meal has been presented (Makkar and Becker, 20108). However, the process is expensive and also not fully disclosed. The feeding value of detoxified jatropha meal for fish was similar to that of soy bean meal.

[0006] Thus, it is worthwhile to address the safety and economic concerns of jatropha oil and seed cakes by attempting to make novel discoveries in detoxification methods and systems to detoxify PEs in a reliable and cost effective manner.

SUMMARY OF THE INVENTION

[0007] The present invention relates to methods and systems for detoxifying phorbol esters in plant products or materials, particularly in plant products or materials from the Euphorbiacaeae family or the Thymelaeaceae family, particularly in the genera Jatropha, Croton, Hippomane, Wikstroemia, Daphne or Pimelea, and more particularly in plant products or materials from Jatropha c rcas, Croton tiglium, Hippomane mancinella, Wikstroemia canescens, Daphne mezereum or Pimelea prostrate.

[0008] In a first aspect, the present invention provides methods for detoxifying phorbol esters in plant products. In accordance with the invention, plant products or materials that contain phorbol esters are irradiated with short wavelength UV light at a radiation intensity and for a period of time sufficient to detoxify or degrade the phorbol esters in the plant products or material. In one embodiment, the plant product or material is oil obtained from the plant or products derived from the oil. In another embodiment, the plant product or material is seed meal or seed cake obtained from the plant after oil crushing or extraction. In a further embodiment, the plant product or material is as disclosed herein. In one embodiment, the plant is a member of the Euphorbiacaeae (examples such as Jatropha curcas, Croton tiglium, Hippomane mancinella) and Thymelaeaceae (Wikstroemia canescens, Daphne mezereum, Pimelea prostrata). In another embodiment, the plant is Jatropha curcas. In another embodiment, the plant is Croton tiglium.

[0009] In one embodiment, the short wavelength UV light has a wavelength from about 100 nm to about 400 nm, preferably from about 100 nm to about 300 nm, more preferably from about 100 nm to about 280 nm, still more preferably from about 100 nm to about 254 nm, and most preferably about 254 nm. In another embodiment, the UV light irradiation level that is useful for treating the oil obtained from the plant or products derived from the oil has an accumulative dosage more than about 0.2 W-sec/cm², preferably more than about 0.4 W-sec/cm², more preferably more than about 0.6 W-sec/cm², more preferably more than about 0.8 W-sec/cm², most preferably from about 1.0 W-sec/cm² to about 1.5 W-sec/cm². In a further embodiment, the UV light irradiation level that is useful for treating the seed meal or seed cake obtained from the plant is from about 6 W-sec/cm², preferably from about 80 W- sec cm², and most preferably from about 90 W-sec/cm² to about 300 W-sec/cm².

[0010] In a second aspect, the present invention provides systems and devices that are suitable for use in the methods of the present invention to detoxify or degrade phorbol esters in plant products or materials. In one aspect, the device or system is designed for inline flowthrough detoxification which is particularly suited for detoxifying oil derived from plants. In one embodiment, the device includes a chamber that houses one or more UV light tubes. In one embodiment, the chamber includes a circular tube which runs the majority of the distance of the chamber and may be internal to the chamber or may be external to the chamber. The circular tube has an inlet and an outlet. In this embodiment, the oil or oil slurry (crushed and blended Jatropha seed kernels with clear solutions such as organic solvents or water, or a slurry soup of Jatropha kernel product is pumped through the circular tube and treated with UV light while being pumped through the circular tube. In another embodiment, the device includes a chamber that houses one or more UV light tubes. The chamber has an inlet and an outlet for flowing oil through the chamber. In this embodiment, the oil is pumped through the chamber and treated with UV light while being pumped through the chamber. In a further embodiment, the system comprises a pipeline and one or more high intensity UV light bulbs. It is understood that more than one high intensity UV light bulb may be used (not shown). At least a portion of the pipeline is transparent so that the high intensity UV light bulbs can treat the oil flowing through the pipeline. In this embodiment, the oil is pumped through the pipeline and treated with UV light from the high intensity UV light bulb while being pumped through the pipeline.

[0011] In second aspect, the device or system is designed for the continuous detoxification of solid plant products or materials. In one embodiment, the system comprises one or more UV light panels and a conveyor belt for transporting the solid plant products past the UV light panels. In this embodiment, the solid plant materials are treated with UV light as they move through, over and/or under the UV light panels. In a third aspect, the device or system is designed for stationary detoxification of solid plant products or materials. In one embodiment, the system comprises a chamber which houses one or more UV light panels and one or more trays for holding the solid plant products. In this embodiment, the solid plant products are treated with UV light in the chamber.

BRIEF DESCRIPTION OF THE FIGURES

[0012] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the leftmost digit(s) of the reference number identifies the drawing in which the reference number first appears.

[0013] Figures 1A and IB show detoxification by PE degradation in Jatropha oil treated by UV (6000 - 7500 uW/cm2, RT) in different time (0 - 3 min). Fig. 1A: Mortality of brine shrimp incubated with oil samples, which were treated by UV light (0 - 3 min). Untreated rapeseed oil was used as the control. Error bars represent the standard deviation from the mean (n = 5). Fig. IB: PE content by HPLC in the same samples.

[0014] Figures 2A-2C show HPLC chromatograms of PE degradation (PEs as indicated by the markings) treated by UV for 0 min (Fig. 2A), 1 min (Fig. 2B) and 3min (Fig. 2C).

[0015] Figures 3A and 3B show detoxification by PE degradation in Jatropha kernel powder treated by UV (21 - 25 mW/cm2, RT) for different lengths of time (0 - 60 min). Fig. 1A: Mortality of brine shrimp incubated with samples that were treated by UV (0 - 60 min). Untreated rapeseed oil was used as the control. Error bars represent the standard deviation from the mean (n = 5). Fig. 3B: PE content by HPLC in the same samples.

[0016] Figures 4A and 4B show dosage dependence of UV inactivation of PE in Jatropha kernel. Fig. 4A: Mortality of brine shrimp incubated with samples treated for 20 min with different UV intensity (21 - 25 mW/cm2 or 34 - 37 mW/cm2, RT). Error bars represent the standard deviation from the mean (n = 5). Fig. 4B: PE content by HPLC in the same samples.

[0017] Figures 4C and 4D show UV inactivation of PE in Croton tiglium oil. Fig. 4C: Mortality of brine shrimp incubated with samples treated for 120 min with UV intensity 28 - 31 mW/cm² at RT. Error bars represent the standard deviation from the mean (n = 5). Fig. 4D: PE content by HPLC in the same samples before (top panel) and after (bottom panel) treatment.

[0018] Figures 5A-5C show different designs for an inline flow system for the detoxification of plant products or materials, particularly oil and seed/kernel slurry derived from the plant, in accordance with the present invention.

[0019] Figures 6A and 6B show a system for continuous detoxification of solid plant products in accordance with the present invention.

[0020] Figures 7A and 7B show a stationary system for detoxification of solid plant products in accordance with the present invention.

[0021] Figure 8 shows that a thinner thickness of seed cake led to more efficient detoxification (at accumulated dosage of 40 W-sec/cm2).

DETAILED DESCRIPTION OF THE ΙΝνΕΝΉΟΝ

[0022] The present invention relates to methods and systems for detoxifying phorbol esters in plant products or materials, particularly in plant products or materials from the Euphorbiacaeae family or the Thymelaeaceae family, particularly in the genera Jatropha, Croton, Hippomane, Wikstroemia, Daphne or Pimelea, and more particularly in plant products or materials from Jatropha curcas, Croton tiglium, Hippomane mancinella, Wikstroemia canescens, Daphne mezereum or Pimelea prostrate.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention belongs.

[0024] The terms "plant product" and "plant material" are used interchangeably herein. These terms are used herein to refer to any part of a plant including crude oil, extracts, whole or defatted kernel meal, processing residues, granulates, grounded particles of any size, leaves, seeds, fruits, sap, branches, ligneous parts, roots and shoots. These terms are also used herein to refer to elements or components derived from a plant, e.g. elements that are modified or processed, like chemical derivates or processed plant oil.

[0025] The term "phorbol ester" as used herein refers to any ester of phorbol, in which two hydroxyl groups on neighboring carbon atoms are esterified to fatty acids. Phorbol and phorbol esters are members of the tigliane family of diterpenes that are defined by polycyclic compounds.

[0026] As used herein, "detoxify" refers to the elimination of all or a substantial portion of the phorbol esters that are initially present in plant products or materials. In accordance with the present invention, this detoxification is accomplished by degrading or destroying the phorbol esters using UV light as described herein.

[0027] As used herein, "a substantial portion" means at least 50%, preferably at least 75%, more preferably at least 90%, still more preferably at least 95%, and most preferably at least 98% of the phorbol ester initially present before treatment is eliminated in the plant products or materials after UV light treatment

[0028] In a first aspect, the present invention provides methods for detoxifying phorbol esters in plant products. In accordance with the invention, plant products or materials that contain phorbol esters are treated with short wavelength UV light at an intensity and for a period of time sufficient to detoxify or degrade the phorbol esters in the plant products or material. In one embodiment, the plant product is oil obtained from the plant or products derived from the oil. In another embodiment, the plant product is seed meal or seed cake obtained from the plant after oil crushing or extraction. In a further embodiment, the plant product or material is as disclosed herein. In one embodiment, the plant is a member of the Euphorbiacaeae family. In another embodiment, the plant is Jatropha curcas. In another embodiment, the plant is Croton tiglium.

[0029] In one embodiment, the short wavelength UV light has a wavelength from about 100 nm to about 400 nm, preferably from about 100 nm to about 300 nm, more preferably from about 100 nm to about 280 nm, still more preferably from about 100 nm to about 254 nm, and most preferably about 254 nm

[0030] In another embodiment, the UV light irradiation level that is useful for treating the oil obtained from the plant or products derived from the oil has an accumulative dosage more than about 0.2 W-sec/cm², preferably more than about 0.4 W-sec/cm², more preferably more than about 0.6 W-sec/cm², more preferably more than about 0.8 W-sec/cm², most preferably from about 1.0 W-sec/cm² to about 1.5 W-sec/cm². [0031] In a further embodiment, the UV light irradiation level that is useful for treating the seed meal or seed cake obtained from the plant is from about 6 W-sec/cm², preferably from about 80 W-sec/cm², and most preferably from about 90 W-sec/cm² to about 300 W-sec/cm².

[0032] In a second aspect, the present invention provides systems and devices that are suitable for use in the methods of the present invention to detoxify or degrade phorbol esters in plant products or materials. In one aspect, the device or system is designed for inline flowthrough detoxification which is particularly suited for detoxifying oil derived from plants. In one embodiment as shown in Fig. 5 A, the device 500 includes a chamber 501 that includes two ends 502a, 502b. The chamber 501 houses two UV light tubes 503a, 503b. Although two UV light tubes are shown in Fig. 5 A, it is understood that one or more UV light tubes may be used . In one embodiment, the chamber 501 includes a winding circular tube 504 which is transparent and runs the distance of the chamber 501 and may be internal to the chamber 501. Alternatively, the winding circular tube 504 may be external to the chamber 501 (not shown). If the winding circular tube 504 is external to the chamber 501, then chamber 501 also needs to be transparent in order for the UV light tubes 503a, 503b to treat the oil. In addition, the winding circular tube 504 does not need to run the entire distance of chamber 501 (not shown). The winding circular tube 504 has an inlet 505 and an outlet 506. In this embodiment, the oil is pumped through the winding circular tube 504 through inlet 505 and treated with UV light from UV light tubes 503a, 503b while being pumped through the winding circular tube 504 and out outlet 506.

[0033] In another embodiment as shown in Fig. 5B, the device 510 includes a chamber 501 that includes two ends 502a, 502b. The chamber 501 houses two UV light tubes 503a, 503b. Although two UV light tubes are shown in Fig. 5B, it is understood that one or more UV light tubes may be used. The chamber 501 has an inlet 505 and an outlet 506 for flowing oil through the chamber 501. In this embodiment, the oil is pumped through the chamber 501 through inlet 505 and treated with UV light from UV light tubes 503a, 503b while being pumped through the chamber 501 and out outlet 506.

[0034] In a further embodiment as illustrated in Fig. 5C, the system 520 comprises a pipeline 521 and a high intensity UV light bulb 522. It is understood that more than one high intensity UV light bulb may be used (not shown). The pipeline 521 has an inlet 523 and an outlet 524 for flowing oil through the pipeline. At least a portion 525 housing high intensity UV light bulb within the pipeline 521 is transparent so that the high intensity UV light bulb 522 can treat the oil flowing through the pipeline 521. In this embodiment, the oil (or product slurry) is pumped through pipeline 521 through inlet 523 and treated with UV light from the high intensity UV light bulb 522 while being pumped through pipeline 521 including transparent portion 525 and out outlet 524.

[0035] In second aspect, the system is designed for the continuous detoxification of solid plant products or materials. In one embodiment as shown in Fig. 6A, the system 600 comprises two UV light panels 601a, 601b and a conveyor belt 602 for transporting solid plant materials 603 past the UV light panels 601a, 601b. It is understood that more than one UV light panel may be used (not shown). The conveyor belt 602 is transparent. One of the UV light panels 601a is positioned below the transparent conveyor belt 602 and the other UV light panel 601b is located in a casing 604 positioned over the transparent conveyor belt 602. The conveyor belt is moved on rollers 605a, 605b, 605c, 605d or any other suitable mechanism (not shown). Solid plant materials 603 is presented on transparent conveyor belt 602 and moved past UV light panels 601a, 601b where the plant materials 603 is treated with UV light. In one embodiment, the solid plant material is a fine powder. As shown in Fig. 6B, a UV light panel 601a comprises a UV tube holder 606 comprising a transparent panel 607 and a UV light tube holder 606. UV light tube holder 606 holds a plurality of UV light tubes 607a, 607b, 607c, 607d, 607e, 607f. In this embodiment, the solid plant materials 603 are treated with UV light as they move over and/or under the UV light panels 601a, 601b.

[0036] In a third aspect, the system is designed for stationary detoxification of solid plant products or materials. In one embodiment as shown in Fig. 7A, the system 700 comprises a chamber 701 which houses a plurality of UV light panels 601a, 601b, 601c, 60 Id and a plurality of holding trays 702a, 702b, 702c for holding the solid plant materials 603. In one embodiment, the solid plant material is a fine powder. Each holding tray, e.g., 702a, is placed between two UV light panels, e.g., 60la and 601b. As shown in Fig. 7B, a holding tray 702a is designed to hold solid plant materials 603. The holding tray 702a is transparent so that the solid plant materials 603 can be treated by UV light. In this embodiment, the solid plant materials 603 are treated with UV light in the chamber 701 from UV light panels 601a, 601b, 601c, 601d.

EXAMPLES

[0037] The present invention is described by reference to the following Examples, which is offered by way of illustration and is not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized. EXAMPLE 1

Materials and Methods

[0038] Materials: Jatropha seeds were collected from a Jatropha Research Farm (JOil Pte Ltd., Singapore). Artemia salina (brine shrimp, INVE of Belgium) cysts were purchased from a local aquatic shop. The natural seawater was filtered using Nalgene 90 mm filter unit (0.45μιη) and its salinity (30-34 parts per thousand, ppt) was measured by a hydrometer (Aquarium Systems, USA). Dimethylsulfoxide (DMSO) and phorbol-12-myristate 13- acetate (PMA) were obtained from Sigma (St Louis, USA). Other chemicals, such as hexane, ethyl acetate, methanol tetrahydrofuran, acid acetic were obtained from Fisher Scientific.

[0039] Crude Oil Extraction from Jatropha Seed Kernels: Jatropha seeds were manually processed with a hand press seed crusher (Piteba, Netherlands). Kernels were separated from coat and ground into fine power in mortar with pestle. After UV treatments, kernel powder was mixed with diatomaceous earth (DE) and the mixture pressed into a IS ml extract cell. Oil extraction was conducted at 105° C at 1200 psi by hexane solvent with an Accelerated Solvent Extractor 200 (ASE 200, Dionex). Re-extraction was conducted in the same method by ethyl acetate with 10% methanol. Solvents were evaporated by nitrogen gas flow at 45° C.

[0040] UV Treatments: Jatropha oil was treated with UV [6000 - 7500 uW/cm² as measured by UV meter (ST-512, Sentry Optronics Corp. at RT)] for different periods of time (from 0 min to 3 min) and 50 ul oil from each sample was used for phorbol esters analysis by HPLC and the remaining oil was mixed with DMSO (1:1 v/v) and stored at -20° C for subsequent toxicity tests. A thin layer (<2mm) of Jatropha kernel power was treated by UV ( 21 - 25 mW/cm² or 34 - 37 mW/cm², RT) for different periods of time ( from 0 min to 60 min) before re-extracted with hexane and ethyl acetate by the Accelerated Solvent Extractor (ACS200, Dionex,) at 105° C, pressure 1200 psi. Hexane or ethyl acetate was evaporated by nitrogen gas flow at 45° C and 50 ul oil from each sample was used for phorbol esters analysis by HPLC and the other oil was mixed with DMSO (1:1 v/v) and stored at -20° C for subsequent toxicity tests.

[0041] Phorbol Esters Analysis by HPLC: 50 ul oil from each sample was dissolved in 200 ul Tetrahydrofuran/Methanol (4:1). PE content was quantified with a HPLC system (Agilent 1200) with Symmetry® C18, 4.6 x 250mm, 5um column (Waters). Mobile phase of water (A) and methanol plus 1% acetic acid (B) was applied. PEs were eluted by a gradient of 60 - 80% B (25min) 80 - 85% B (15min) then hold at 95% B (lOmin). External standard PMA (5 mg ml) was serially diluted (2mg/ml, 1 mg/ml, 0.5mg/ml and 0.25mg/ml) in methanol and 1 ul solution was injected and analyzed by HPLC at 280 nm by a DAD detector. Average peak areas of three replicates were used to build a standard curve of good linearity (R² > 99%) that was used to quantify total PE content in samples, which appeared in five peaks between 31 and 35 min.

[0042] Brine Shrimp Bioassay: 100 mg of brine shrimp cysts were incubated in natural seawater in a petri dish at 25 °C, with indoor lighting for 24 h. Newly hatched nauplii were transferred into another petri dish with fresh seawater and maintained for another 24 h. After a total incubation time of 48 h, 50 μΐ seawater containing ten 48 h old live brine shrimps were pipetted to each well in a 24- well tissue culture plate, flat bottom with low evaporation lid (Falcon) and topped up with 400 μΐ of fresh seawater, making up to a volume of 450 μΐ. For each treatment, six wells with 60 nauplii in total were used for each toxicity testing. 50 ul equal volume mixture of DMSO with re-extracted oil after each treatment was then added into each well making final volumes of 500 μΐ each. These plates were then further incubated for another 24 h without feeding. 50 ul equal volume mixture of rapeseed oil with DMSO was used as the negative control. Number of surviving (free moving or moving upon stimulation) nauplii was counted at the end of 24 h using a stereo microscope (Leica). Mortality ratio was calculated as percentage of death in 60 nauplii. Toxicity bioassay for each treatment was repeated four more times.

EXAMPLE 2

Detoxification of Phorbol Esters by UV in Jatropha Oil

[0043] After crude Jatropha oil samples were exposed to UV light (6000 - 7500

RT) for various durations (from 0 min to 3 min), Jatropha oil was analyzed for PE content and toxicity to brine shrimp. Concentration of exposed and unexposed oil samples in the bioassay system were 5% of final volumes. As shown in Fig 1 A, at 0 min, 100% mortality of brine shrimp was observed. For UV treatment, oil samples that were incubated for 1 min, mortality dropped significantly to less than 15%, and more so to less than 3% at 3 min sample, similar to that for rapeseed oil control.

[0044] The oil samples were subjected to HPLC analysis for PE content. As shown in Fig IB, a similar pattern of PE degradation was observed with UV treatment oil samples: within 1 min, PE level fell about 20% of that for 0 min and became undetectable within 3 min. Figure 2 shows some HPLC chromatographs. In comparison with Jatropha without UV treatment (0 min, Figure 2A), the peak area for PE was significantly decreased for sample after 1 min (Figure 2B) and non-detected after 3 min of UV treatment (Figure 2C). I I

EXAMPLE 3

Detoxification of Phorbol Esters by UV in Jatropha Kernel

[0045] After crude Jatropha kernel samples were exposed to UV light (21 - 25 mW/cm², RT) for various durations (from 0 min to 60 min), Jatropha oil was re-extracted with hexane and ethyl acetate by Accelerated solvent extractor (ACS200, Dionex^ at 105° C, pressure 1200 psi. Hexane or ethyl acetate was evaporated by nitrogen gas flow at 45° C and remaining oil was mixed with DMSO (1:1 v/v) and stored at -20° C for subsequent toxicity tests. Jatropha oil was analyzed for PE content and toxicity to brine shrimp. Final concentration of oil in the bioassay system was 5%. As shown in Fig 3A, at 0 min, 100% mortality of brine shrimp was observed. For UV treatment samples, within 5 min, mortality dropped to about 70%, and so to less than 15% at 60 min.

[0046] The oil samples were subjected to HPLC analysis for PE content. As shown in Fig 3B, a similar pattern of PE degradation was observed with UV treatment oil samples: within 5 min, PE level fell about 55% of that for 0 min and about 20% of that for 0 min within 60 min.

EXAMPLE 4

Dosage Dependence of UV Inactivation of Phorbol Esters in Jatropha Kernel

[0047] We further tested if such degradation detoxification depended on dosage of UV. We use two UV intensities, 21 - 25 mW/cm² and 34 - 37 mW/cm², to treat the Jatropha kernel for 20 min. As shown in Fig 4A, for kernel treated with 21 - 25 mW/cm² of UV for 20 min, about 55% mortality of brine shrimp were observed. In contrast, after exposure to 34 - 37 mW/cm² for the same 20 min, mortality of brine shrimp dropped significantly to 20%. Fig 4B shows the PE content by HPLC for the samples treated by different UV intensity (21 - 25 mW/cm² or 34 - 37 mW/cm²). This result suggests that the PE degradation/detoxification depends on dosage of UV.

EXAMPLE 5

Dosage Dependence of UV Inactivation of Phorbol Esters in Croton tiglium oil

[0048] We further tested if such degradation detoxification by UV irradiation happens to other PE containing products derived from other plants. Croton tiglium oil was purchased from Sigma and subjected to UV irradiation (28 - 31 mW/cm², RT ) for 120 min. As shown in Fig 4C, oil treated with 28 - 31 mW/cm² of UV for 120 min let to only 20% mortality for brine shrimp. Untreated rapeseed oil was used as the control. Error bars represent the standard deviation from the mean ( n = 5 ). Fig 4D shows the PE content by HPLC for the sample to be less than 10% of that of the untreated sample. This result suggests that the PE degradation detoxification by UV can happen to any PE containing plant product.

EXAMPLE 6

Relationship Between Thickness of Seed Cake and Efficiency of Detoxification

[0049] We further tested relationship between seed cake thickness and efficiency of detoxification. We built a custom made stainless steel UV chamber installed with five parallel arranged 15W UV tubes which providing an intensity of 22 mW/cm² at distance of 8 cm. Seed kernel powder were spread evenly on glass plates at different thickness (0.75 mm to 3 mm) before UV illuminating for 30 min in chamber before analysis of remaining PE quantity. As shown in Figure 8, thickness of seed cake is negatively related to efficiency of detoxification, mostly due to the limited penetration by short wave UV. For seedcake of 0.75 mm thickness, 30 min UV treatment (accumulated dosage of about 40 W-sec / cm²) would eliminate 70% of PEs whereas increasing thickness had lower efficiency of detoxification.

Discussion

[0050] Degradation of Phorbol Esters: In Jatropha, curcin and PEs are the major toxic components. Curcin, being a protein, can be easily degraded in soil and is unlikely to exhibit significant toxicity under in vivo conditions (Lin et al., 2003; Gressel, 2008; Achteri et al., 2008; King et al., 2009). Moreover, it does not go into oil and can be easily inactivated by heat treatment of seed cake. Hence it does not pose major safety problem. In comparison, PEs are hydrophobic, present and heat stable in both oil and seed cake/meal (Makkar et al., 2008a). During the process of oil extraction from Jatropha seeds, 70-75% of PEs are extracted along with the oil and 25-30% of PEs still remain strongly bound to the matrix of seed meal (Makkar et al., 2008b; Makkar et al., 2009). Due to this toxicity of PEs, seed cake cannot be used in animal feeds without removing PEs. Although nutrient-rich seed cake is suitable as fertilizers (Francis et al., 2005; Achten et al., 2008), there are also concerns over the application of seed cake as fertilizers due to its potential toxicity associated with PEs leaching, safe disposal and its impact on beneficial microbial communities, insects, invertebrates and plant/animal communities (Gressel, 2008; Achten et al., 2008).

[0051] Degradation of soil phytochemicals and biochemicals is generally through microbial action, or by photodegradation. A recent research by Devappa et al. have reported that PEs present either in Jatropha oil or Jatropha seed cake are degradable in soil over a period of time in the dark, mostly through microbial activities. Such degradation of PEs increases with an increase in temperature and moisture content in the soil (Devappa et al., 2010b). The incubation of PE with soil was conducted in the dark in order to "minimize the effect of light". In our recent research, we found that sunlight can degrade and detoxify PE within days. Such detoxification is independent of the presence of soil, types of soil or the presence of microorganisms (data not shown). Detoxification by sunlight, however, lacks industry applicability for its requirement of sunlight and exposure time required. We turned to short wave UV, which has been used widely for disinfection, decontamination of surfaces, water and air. We exposed the Jatropha oil and kernel to UV. The results showed that PE was degradable after exposure to UV. All these point to the conclusion that light can act directly on PE structure, break its structural integrity and reduce bioacivity. As long as PE containing oil or kernel is exposed to UV, UV induced degradation can effectively breaks and detoxify PEs. With two complementary mechanisms of degradation and detoxification of PE in working, it is highly unlikely that PE will remain intact and toxic under UV conditions.

[0052] Simple and Effective PE Toxicity Bioassay with Brine Shrimp (Anemia salina) System: Carp has been used in bioassay of phorbol esters due to its high susceptible to phorbol esters present in Jatropha. The threshold level at which phorbol esters cause adverse effect was 15 ppm in the diet (Becker and Makkar, 1998). Fresh water snails are sensitive to PEs and also used in bioassays. Compared to aqueous extracts, methanol extract showed much higher toxicity against all tested organisms (Goel et al., 2007). Methanol extracted PEs was also used in recent study of PE degradation in the absence of light (Devappa et al., 2010b). Brine shrimp was chosen for its easy availability and good ecological relevance (bottom consumer in the Primary food chain). Five percent of Jatropha oil present in water (equivalent to 12 mg L of PE) was enough to kill all nauplii within 24 hours (data not shown). This showed the good sensitivity of the system. We compared 24 h (instar I) with 48 h (instar ΙΙ-ΠΙ) Artemia salina nauplii and found 48 h old nauplii more sensitive to PE due to active filter feeding which is made possible with a developed digestive tract only from stage instar Π onwards (data not shown). This observation was in line with several papers, which had shown that instar ΙΙ-ΠΙ nauplii to be the most sensitive stage for most tested toxins compared to other age classes (Sorgeloos et al., 1978; Vanhaecke et al., 1981; Barahona and Sanchez-Fortun, 1996). Nauplii older than 48 h did not have increased sensitivity and may require active feeding which may complicate results. Further optimization was using DMSO, a polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. Using DMSO effectively solves the problem of insolubility of PEs in water. Testing with edible and non-toxic rapeseed oil, the system with 5% final concentration of DMSO has very low residue toxicity to brine shrimp (< 2%).

[0053] Phorbol esters are naturally produced by plants of the Euphorbiacaeae (examples such as Jatropha curcas, Croton tiglium, Hippomane mancinella) and Thymelaeaceae (Wikstroemia canescens, Daphne mezereum, Pimelea prostrata). Croton tiglium, known as Purging Croton, is a plant species in the Euphorbiaceae family. It is one of the 50 fundamental herbs of used in traditional Chinese medicine, where it has the name Ba Dou. Phorbol esters were firstly isolated from Croton tiglium oil in 1934 where they are present in high concentrations. We also tested UV irradiation on commercially available croton oil, PEs were effectively degraded (reduced to <10% of the untreated sample) and toxicity was reduced significantly to about 20% mortality in brine shrimp bioassay model. This test effectively exemplifies that any PE containing plant product can be detoxicated with the same type of UV treatment.

[0054] Another feature of our system is to cultivate small number (10) of brine shrimps in small wells (volume of 500 ul) and to count dead shrimp with a stereo microscope. That greatly increases efficiency and reduces possible miscounting that often occurs for higher culture density in large volumes, and also reduce errors by introducing variables such as aeration, water quality, etc. An important advantage of brine shrimp is the dosage dependent response to PE (1% - 4%, equivalent to PE 25 mg/L - 100 mg/L correlate with 5% - 95% of mortality in a linear manner) (Bu et al., 2012). Our brine shrimp bioassay system is quite comparable to other bioassay systems. It can be easily adapted for routine monitoring of PE toxicity present in Jatropha oil or water.

[0055] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0056] It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

BIBLIOGRAPHY

[0057] Achten, W.M.J. et al. (2008). "Jatropha bio-diesel production and use." Biomass and Bioenergy 32(12):1063-1084.

[0058] Barahona, M.V. and Sanchez-Fortun, S (1996). "Comparative sensitivity of three age classes of Artemia salina larvae to several phenolic compounds." Bull Environ Contam Toxicol 56:271-278.

[0059] Becker, K. and Makkar, H.P.S. (1998). "Effects of phorbol esters in carp (Cyprinus carpio L)." Veterinary and Human Toxicology 40(2):82-86.

[0060] Bu, Y.P. et al. (2012). "Light induced degradation of phorbol esters." Ecotoxicol Environ Saf 84:268-273.

[0061] Devappa, R.K. et al. (2008). "Biochemical and nutritional evaluation of Jatropha protein isolate prepared by steam injection heating for reduction of toxic antinutritional factors." J Sci Food Agric 88:911 -919.

[0062] Devappa, R.K. et al. (2010a). "Nutritional, biochemical, and pharmaceutical potential of proteins and peptides from jatropha: review." J Agric Food Chem 58(11):6543-55.

[0063] Devappa, R.K. et al. (2010b). "Biodegradation of Jatropha curcas phorbol esters in soil." J Sci Food Agric 90:2090-2097. [0064] Francis, G. et al. (2005). "A concept for simultaneous wasteland reclamation, fuel production, and socio-economic development in degraded areas in India: Need, potential and perspectives of Jatropha plantations." Natural Resources Forum 29(l):12-24.

[0065] Gross, H. et al. (1997). "Detoxification of J. Curcas press cake and oil and feeding experiments in fish and mice." In Biofuels and Industrial Products from Jatropha curcas, Giibitz, G.M. et al., Eds., Dbv, Graz, pp.179-182.

[0066] Haas, W. And ittelbach, M. (2000). "Detoxification experiments with the seed oil from Jatropha curcas L." Industrial Crops Products 12:111-118.

[0067] GEXSI (2008). Global Market Study on Jatropha.

[0068] Goel, G. et al. (2007). "Phorbol esters: Structure, biological activity, and toxicity in animals." International Journal of Toxicology 26(4):279-288.

[0069] Gressel, J. (2008). "Transgenics are imperative for biofuel crops." Plant Science 174(3):246-263.

[0070] Gubitz, G.M. et al. (1999). "Exploitation of the tropical oil seed plant Jatropha curcas L." Bioresource Technology 67(l):73-82.

[0071] Haas, W. et al. (2002). "Novel 12-deoxy-16-hydroxyphorbol diesters isolated from the seed oil of Jatropha curcas." Journal of Natural Products 65(10): 1434-1440.

[0072] King, A J. et al. (2009). " Potential of Jatropha curcas as a source of renewable oil and animal feed." J Exp Bot 60: 2897-2905.

[0073] Kumar, H.P.S. et al. (2008). "Detoxification of Jatropha curcas seed meal and its utilization as a protein source in fish diet." Comp Biochem Physiol 151A: 13-14.

[0074] Lin, J. et al. (2003). "Antitumor effects of curcin from seeds of Jatropha curcas." Acta Pharmacologica Sinica 24(3):241-246.

[0075] Makkar, H.P.S. and Becker, K. (1997). "Jatropha curcas toxicity: identification of toxic principle(s)." In Toxin Plants and other Natural Toxicants. T. Barland and A. C. Barr, Eds., CAB International, New York, pp. 554-558.

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[0078] Makkar, H.P.S. et al. (2008b). "Variations in seed number per fruit, seed physical parameters and contents oil, protein and phorbol ester in toxic and non-toxic genotypes of Jatropha curcas." J Plant Science 3:260-265.

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[0081] Sorgeloos, P. et al. (1978). "The use of Artemia nauplii for toxicity tests--A critical analysis." Ecotoxicol Environ Saf 2:249-255. [0082] Vanhaecke, P. et al. (1981). "Proposal for a short-term toxicity test with Artemia nauplii." Ecotoxicol Environ Sa/5:382-387.

Claims

WHAT IS CLAIMED IS:

1. A method for detoxifying phorbol esters in plant products or materials containing phorbol esters which comprises treating the plant products or materials with short wavelength UV light irradiation sufficient to detoxify or degrade the phorbol esters in the plant products or material.

2. The method of claim 1, wherein the plant material is oil obtained from plants or products or materials derived from the oil.

3. The method of claim 1, wherein the plant product or material is seed meal or seed cake obtained from the plant after oil crushing or extraction.

4. The method of any one of claims 1-3, wherein the short wavelength UV light has a wavelength from about 100 ran to about 400 nm, preferably from about 100 ran to about 300 nm, more preferably from about 100 nm to about 280 nm, still more preferably from about 100 nm to about 254 nm, and most preferably about 254 nm.

5. The method of any one of claims 1-4, wherein the UV light irradiation level that is useful for treating the oil obtained from the plant or products derived from the oil has an accumulative dosage more than about 0.2 W-sec/cm², preferably more than about 0.4 W-sec/cm², more preferably more than about 0.6 W-sec/cm², more preferably more than about 0.8 W-sec/cm², most preferably from about 1.0 W-sec/cm² to about 1.5 W-sec/cm².

6. The method of any one of claims 1-5, wherein the UV light irradiation level that is useful for treating the seed meal or seed cake obtained from the plant is from about 6 W-sec/cm², preferably from about 80 W-sec/cm², and most preferably from about 90 W-sec/cm² to about 300 W-sec/cm².

7. The method of any one of claims 1-6, wherein the plant is a member of the Euphorbiacaeae family or a member of the Thymelaeaceae family.

The method of claim 7, wherein the plant is a member of the genera Jatropha, Croton, Hippomane, Wikstroemia, Daphne or Pimelea.

The method of 8, wherein the plant is Jatropha curcas, Croton tigli m, Hippomane mancineUa, Wikstroemia canescens, Daphne mezereum or Pimelea prostrate.

A system for inline flowthrough detoxification of plant products or materials containing phorbol esters which comprises

a chamber that houses one or more UV light tubes and

a circular tube having an inlet and an outlet, wherein the tube runs the majority of the . distance of the chamber and wherein the tube is internal to the chamber or external to the chamber.

a chamber that houses one or more UV light tubes that has an inlet and an outlet for flowing plant products or materials through the chamber.

a pipeline having at least a portion of which is transparent and

one or more high intensity UV light bulbs.

The system of any one of claims 10-12, wherein the plant material is oil obtained from plants or products or materials derived from the oil.

A system for the continuous detoxification of solid plant products or materials containing phorbol esters which comprises

one or more UV light panels and

a conveyor belt for transporting the solid plant products past the UV light panels.

A system for stationary detoxification of solid plant products or materials containing phorbol esters which comprises a chamber comprising two or more UV light panels and one or more trays for holding the solid plant products or materials.

16. The system of claim 14 or 15, wherein the plant product or material is seed meal, seed cake, kernel or kernel slurry obtained from the plant after oil crushing or extraction.

17. The system of any one of claims 10-16, wherein the UV light is short wavelength UV light having a wavelength from about 100 nm to about 400 run, preferably from about 100 nm to about 300 nm, more preferably from about 100 nm to about 280 nm, still more preferably from about 100 nm to about 254 nm, and most preferably about 254 nm.

18. The system of any one of claims 10-17, wherein the UV light irradiation level that is useful for treating the oil obtained from the plant or products derived from the oil has an accumulative dosage more than about 0.2 W-sec/cm², preferably more than about 0.4 W-sec/cm², more preferably more than about 0.6 W-sec/cm², more preferably more than about 0.8 W-sec/cm², most preferably from about 1.0 W-sec/cm² to about 1.5 W-sec/cm².

19. The system of any one of claims 10-17, wherein the UV light irradiation level that is useful for treating the seed meal or seed cake obtained from the plant is from about 6 W-sec/cm², preferably from about 80 W-sec/cm², and most preferably from about 90 W-sec/cm² to about 300 W-sec/cm².

20. The method of claim 1, wherein the UV light treatment is performed using the system of any one of claims 10-19.