The Production Processes Of Resveratrol Biology Essay

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Plants are synthesizing trans-resveratrol when it was infected by microbes or exposed to ultraviolet (UV) radiation. Resveratrol is produced in response to injury and stress. Resveratrol is classified as phytoalexin because of its production from grapes through responses to biotic and abiotic stresses. Phytoalexin is a class of compounds, including resveratrol which have antibiotic properties that are naturally produced by some plants to fight infections from the fungus. Phytoalexin belong to stilbene family which are derivatives of the trans-resveratrol structure (3,5,4'-trihydroxystilbene). Resveratrol is synthesized from phenylalanine via the phenylalanine or polymalonate pathway. It is converted into cinnamic acid by phenylalanine ammonia lyase (PAL). In the final step the stepwise condensation of three molecules of malonyl-CoA is catalysed by stilbene synthase as shown in the figure 13 (Kim Trollope, 2006). Reaction that produces resveratrol in the plant is similar to a reaction using the same products catalyzed by a similar enzyme which is chalcone synthase (Goodwin et al., 2000). Chalcone synthase combines p-coumaroyl CoA and malonyl CoA to form chalcones including naringenin and eriodictyol which go on to form flavonoids which are responsible for the anthocyanins. Anthocyanins are a class of compounds that include the pigments in grapes (Croteau et al., 2000).

Figure 13: Biosynthesis of resveratrol from phenylalanine via phenylpropanoid pathway (Becker et al., 2006).

Resveratrol is produced at the abaxial surface of the leaves and skin of the grapes. The seeds are lesser extent. The production of resveratrol in the skin is higher concentration. Fresh grape skin contains 50.0g/g to 100 0g/g (0.22 0mol/g to 0.44 0mol/g) of trans-resveratrol (Hendler & Rorvik, 2001). As they ripen the skin of the grapes, grapes produce less resveratrol. Cis-Resveratrol is found in wine but contain lower levels than the trans-resveratrol. These forms were created during the winemaking process. The most important factor that will affect the level of resveratrol is the length of time the skin is kept with the grape must during the winemaking process. The longer times will increase resveratrol concentration. While in white wine production, the skin is always removed for fermenting, giving these wines a lower resveratrol concentration than red wines. Rose wine is a combination of red and white wines have an intermediate concentration. Commercial producers of resveratrol induce plants to produce greater quantities when adding aluminum chloride or aluminum sulfate to grape shoots and vines. This will help to produce greater quantity. Production of resveratrol in harvested grapes increased twofold with irradiation by UVB light and threefold with irradiation by UVC light (Cantos et al., 2000).

Grape plants excreted trans-resveratrol from leaves' wounds touching a cellulosic substratum, such as filter paper and soaked with inducers in aqueous solution. These inducers are

monosaccharides, disaccharides, some polysaccharides and Cu2+ ions. This inducer can help to increase the level of resveratrol. Resveratrol can be extracted from these plants with water and alcohol or with methanol and ethyl acetate (Vastano et al., 2000).

2.4.1 Ultrasonication-assisted extraction method of resveratrol from grapes

Yong-Jin Cho and friends in 2005 had done a research on ultrasonication-assisted extraction method of resveratrol from grapes, an effective extraction method of resveratrol from grapes. Ultrasonication assisted extraction is the disintegration of cell structures (lysis) which means used ultrasound for the extraction of intra-cellular compounds. Stem in grape may also be a source of resveratrol rather than just its fruit. The contents of resveratrol in fruit stem were 170-440μg/g-dry material while those in skin and seed were 4-8μg/g-dry material. The flesh of the grapes was hardly detected. Ethanol is being mainly utilized due to the use of resveratrol as the efficiency of extraction. Ultrasonication-assisted extraction was used to obtain resveratrol. The highest yield of resveratrol was obtained with ethanol/water 80:20% (v/v). The result of ultrasonication-assisted extraction at a room temperature was very effective. The grape stems were used as a resource of resveratrol in the ultrasonication-assisted extraction method. The Campbell and Gerbong grapes were selected. A solvent extraction system was run at room temperature. An ethanol/water 80:20% (v/v) solution was used as a solvent. The ratio of sample weight to solvent volume was 8g/l. While the ultrasonic power with the frequency of 47kHz. After extraction by ultrasonication-assisted method, insoluble materials were removed by centrifugation for 15min. Finally, extracts were concentrated by evaporation.

The analysis the content of resveratrol in the extract was determined by the HPLC system. The extract was solved in methanol and the solution of 20μl was injected. The elution profile was acetonitril/water 40:60% (v/v) for 8min, acetonitril/water 10:90% (v/v) for 27min, and acetonitril/water 40:60% (v/v) for 15min under the flow rate of 0.6ml/min. The peak for resveratrol was detected was showed in the figure 12. Figure 14 showed the result of HPLC chromatogram of resveratrol 99% grapes skin extract.

As shown in Table 4, the ultrasonication-assisted extraction is effective for extracting resveratrol in faster rate. Similar to the traditional solvent extraction, the ultrasonication-assisted extraction had effective extraction time because longer duration might cause the degradation of solubilized resveratrol. Traditional solvent extraction takes longer duration. Measured and predicted amounts (μg/g-dry material) of resveratrol extracted from fruit stem of Campbell grape Gerbong grape with respect to time when the ultrasonication-assisted extraction was applied is shown in figure 15 and figure 16. The ultrasonication-assisted extraction of resveratrol in this study was considered to be very effective. Extraction of resveratrol was applied to fruit stem of Campbell and Gerbong grapes, the recovery yield was increased by 24-30%, compared to that by the conventional solvent extraction. As for fruit stem of Campbell grape, the amount of resveratrol solubilized by the ultrasonication-assisted extraction method for 3min was 438μg/g-dry material, which was higher than 353μg/g-dry material by the traditional solvent extraction with the condition of 60°C and 30min. As for fruit stem of Gerbong grape, the recovery with 171μg/g-dry material, by the new method for 10min was increased compared to that with 131μg/g-dry materials by the traditional method. Fruit stem contains more resveratrol, a stilbenoid phytoalexin, which will be used as an antioxidant and anticancer compound, than other parts such as fruit skin and seed in grapes (Yong Jin Cho et al., 2005).

Table 4: The recoveries by the ultrasonication-assisted extraction for 15min for fruit stem of Campbell grape and Gerbong grape

Raw material

Extraction time (min)

Resveratrol content (μg/g dry material)

Relative recovery

Fruit stem of Campbell grape

1

354

1.003

3

438

1.241

5

413

1.170

10

397

1.125

15

279

0.790

Fruit stem of Gerbong grape

1

123

0.939

3

158

1.206

5

166

1.267

10

171

1.305

15

167

1.275

(Yong Jin Cho et al., 2005).

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Figure 14: HPLC chromatogram of resveratrol 99% grapes skin extract (Yong Jin Cho et al., 2005).

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Figure 15: Measured and predicted amounts (μg/g-dry material) of resveratrol extracted from fruit stem of Campbell grape with respect to time when the ultrasonication-assisted extraction was applied. (Yong Jin Cho et al., 2005).

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Figure 16: Measured and predicted amounts (μg/g-dry material) of resveratrol extracted from fruit stem of Gerbong grape with respect to time when the ultrasonication-assisted extraction was applied. (Yong Jin Cho et al., 2005).

2.4.2 Extraction method of resveratrol from muscadine juice

Muscadine grapes are a species in the southeastern United States. It can be extracted using either hot press or a cold press method. The difference is whether the crushed grapes should heat before pressing. Hot pressed juice has higher titratable acidity, more juice yields and greater color extraction than cold press juice of the same cultivar. Both hot press and cold press processes use pectic enzymes to improve juice extraction by breaking down pectins that trap juice in the tissue (Morris and Brady, 2004). The enzyme may also affect the extraction of resveratrol from the fruit tissue from 'Noble' and 'Carlos' cultivars of muscadine grapes that was collected from the USDA Small Fruit Crops laboratory in Poplarville, MS. 'Carlos' is a bronze skinned grape. 'Noble' is a black skinned grape. Both also used for juice and wine production. Three replicates were harvested for each cultivar. After harvest, samples were refrigerated during transport. Besides two cultivars of muscadine grapes, two cultivars of bunch grapes (Vitis labrusca, 'Midsouth' and 'Miss Blue') were collected from the Hill Farm in Baton Rouge, LA. 'Midsouth' is a purple bunch grape and 'Miss Blanc' is a white bunch grape. Three replicates were taken for each cultivar. All samples were crushed using a manually de-stemmer or operated crusher. First sample of grapes was crush in the process of fresh press. After crushing, a 20 ml sample was collected from the juice that ran freely from the crushed fruit. After collecting the free run sample, the remaining fruit were pressed using a hydraulic rack and frame press. A 20 ml sample was collected from the resulting juice. The next group sample of grapes were crushed and quickly heated in a steam kettle to about 60 °C and quickly pressed with hot pressed process. A 20 ml sample was collected from the resulting juice. Hot pressed juice had higher total phenols, darker color and greater acid extraction in muscadine grapes. Hot press method used to extract dark grape juice. For the next group of grapes were placed in a sealed plastic bag and frozen over night at -20°C. After freezing, the grapes were thawed and crushed. A 20 ml sample was collected from the resulting juice. Freezing method resulted in higher juice stilbene concentration in muscadine juice. Freezing method causes the breakdown of cell membranes and cell walls that allow stilbenes to become dissolved in the juice. While for the last group of grapes were crushed and placed in a two liter container. Pectic enzyme was stirred into the crushed grapes at a concentration of 100 ppm by weight. Then the sample was placed in cold storage at 45 °F overnight. After enzyme treatment, the crushed fruit were pressed. A 20 ml sample was collected from the resulting juice (Figure 17). Pectic enzymes used to increase the amount of juice extracted from fruit. Pectic enzymes will breakdown the pectins that are present in the juice tissue of the grape. When adding pectic enzymes before pressing, juice that trapped in the tissue can be released. For all juice samples, soluble solids was measured using a digital refractometer (Bellingham and Stanley, model RFM 80). Juice yield data were collected for cold press, hot press, frozen and enzyme treatments. All juice samples were frozen at -40°C until HPLC analysis. All juice samples were thawed and centrifuged to remove particulate matter. A sample of the supernatant was filtered through 0.2 μm Nucleopore Track-Etch membrane (polycarbonate) in preparation for HPLC analysis. After microfiltration, samples were placed in amber auto sampler vials to protect them from light induced isomerization. Sample analysis was done using HPLC with a UV detector. The analytical equipment consist of a Waters 600 pump, a Waters 717 plus auto sampler, a Waters 2487 dual wave length UV detector. The column was a Waters Sun fire 3 x 250 mm C18 (5 μm particle size) with a 20 mm pre-column of the same material. Column temperature was maintained at 30 °C. Each sample contains 20 μL was injected and eluted using an isocratic method with a mobile phase of 69.3 (water):22 (acetonitril):8 (propanol):0.7 (formic acid) by volume at a flow rate of 0.2 ml/min. Samples were analyzed by a UV detector at 285 nm and 306 nm for trans and cis isomers of resveratrol and piceid (Careri et al., 2003). There is no standards have cis isomers of resveratrol. Standards of both trans resveratrol were exposed to direct sunlight for approximately 15 minutes to achieve cis compounds in the samples. This resulted conversion of about 85% of the trans compounds to cis isomers and was confirmed by HPLC analysis of the standard before and after exposure to sunlight (Mark R. LeBlanc, 2006). Pectic enzyme treatment increased 'Carlos' juice yield from 41.5 % to 61.1 % and 'Noble' juice yield from 40.8% to 56.2%. Pectic enzyme treatment resulted in increase juice yield compare to all other treatments. Even though enzyme treatment produced greatest juice yield, but did not result in the greatest resveratrol concentration. The hot press treatment had the greatest resveratrol levels. The reason is the increase in juice yield resulting from the pectic enzyme treatment comes from the pulp. The muscadine fruit pulp has the lowest stilbene concentration because the additional juice extracted from the pulp may dilute the resveratrol concentration of the juice in the enzyme treatment. The hot press treatment extracting resveratrol from the skin, therefore hot press treatment has higher resveratrol concentration than enzyme treatment. Majority of the resveratrol in the fruit is located in the skins. The skins of the bunch grapes are thinner than those of muscadine skins, the resveratrol concentration is higher. Resveratrol more easily extracted from the thin skins of bunch grapes than the thick skins of the muscadine grapes. 'Miss Blue' and 'Midsouth' would produce a juice with greater resveratrol concentration than 'Carlos' and 'Noble' muscadine grapes (Mark R. LeBlanc, 2006).

Figure 17: Juice extraction methods. (Mark R. LeBlanc, 2006)

2.4.3 Supercritical fluid extraction of piceid, resveratrol and emodin from Japanese knotweed

Extraction method with supercritical fluids is extraction technique that can give good yields of clean extracts. This technique is similar to classical Soxhlet extraction, but the solvent used is a supercritical fluid, a substance above critical temperature and pressure. Supercritical fluid extraction (SFE) is an extraction technique that sample was used with analysis polyphenolic compounds from plant matrices. SFE isolation and determine resveratrol from grape skin of Vitis vinifera. The herbal extracts obtained from the fresh of the dried plants, or parts of plants like roots, leaves, flowers or seeds. Polyphenolic compounds found in plants have multiple biological effects, includes antioxidant. Japanese knotweed is a kind of chinese traditional medicinal herb used for the treatment of various diseases. Resveratrol (3,4′,5-trihydroxystilbene) and piceid (3,4′,5-trihydroxystilbene-3-β-d-glucoside) is a stilbenoid glucoside and is a major resveratrol derivative in grape juices were isolated from the extracts prepared from the roots of Japanese knotweed. Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) is anthraquinone based compound which is abundantly represented in Japanese knotweed. Samples of a plant Japanese knotweed were investigated in the study. The milled powder of the dried roots was obtained. The powder was homogeneous. The particle size was less than 0.5mm.

Supercritical fluid extractions (SFE) were performed on a SE-1 instrument. The effects of different parameters like type of organic modifier, temperature, extraction time and pressure were evaluated. The pressure was at between 40 MPa, temperature at 100 C and extraction time 45min were chosen because of isolation of more polar compounds from plant matrices. All extractions were performed with modified carbon dioxide in dynamic mode. The effects of organic modifier were studied. Tested modifiers were methanol, ethanol and acetonitrile. Amounts of analytes in 1g extracted sample obtained with the modifiers are shown in figure 18. As shown from the bar chart, the best results achieved when acetonitrile was used showed the best result, the highest substance content was achieved while acetonitrile was used as modifier. While for pressure, four different pressures like 25, 30, 35 and 40MPa were tested using the fixed conditions as temperature 100 C, 45min of extraction time and modifier acetonitrile. Pressures lower than 25MPa were not tested because of the poor extraction efficiency for more polar compounds. High pressure causes higher extraction yields of all target compounds. Figure 19 showed extraction efficiency on pressure applied. The pressure increase causes the increase of piceid and resveratrol content. The pressure of 40MPa was chosen as the most suitable for extraction of all three substances. For the effect of temperature, tested temperature range was between 50 to 110°C under constant pressure, extraction time was 45min and acetonitrile as modifier. As figure 20 showed, the increase efficiency in dependence on increasing temperature. The highest substance content was achieved when applied temperature at 100 C. While at 110 C a slight decrease of piceid and emodin content was observed. The temperature 100 C was chosen as suitable for further experiments. While the effect of extraction time was investigate using the fixed condition of 40MPa, 100°C and acetonitrile as modifier. Six various lengths of the extraction were tested which were 10, 15, 20, 30, 45 and 60min. Figure 21 showed the extraction efficiency on the extraction time. Bar charts showed the increase of substance contents in the length of extraction 45min. Thus, 45min was chosen as extraction time for extraction of all target compounds (Blanka Benova et al., 2009).

image

Figure 18: Effect of modifier on extraction efficiency of analyzed compounds (extraction conditions: 40MPa, 100 C, 45min). (Blanka Benova et al., 2009).

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Figure 19: Effect of pressure on extraction efficiency of analyzed compounds (extraction conditions: 100 C, 30min, and modifier acetonitrile). (Blanka Benova et al., 2009).

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Figure 20: Effect of temperature on extraction efficiency of analyzed compounds (extraction conditions: 40MPa, 30min, and modifier acetonitrile). (Blanka Benova et al., 2009).

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Figure 21: Effect of extraction time on extraction efficiency of analysed compounds (extraction conditions: 40MPa, 100°C, and modifier acetonitrile). (Blanka Benova et al., 2009).

Supercritical CO2 continually pumped through the extraction vessel. The modifier 5% (v/v), fixed level based on the loop size was added through the 500μL loop in static mode during the pump filling step. The stainless steel extraction vessel was packed with 1g of sample and glass beads were added to fill in the remaining volume of the extraction cell. A fused silica tube restrictor with inner diameter 50μm and length 20cm was used to transfer extracted analytes into the collection unit. The restrictor outlet heated to prevent plugging and immersed in a vial with 5mL of methanol based on the construction of the collection unit, which allows the extract entrapment into the collection solvent.

While the Soxhlet extraction was performed as 1g of the sample was inserted into the cellulose extraction cartridge together with cotton-wool. This is to prevent washing out the sample and extracted with 70mL of solvent under reflux for 4 hours. After the extraction finished, the solvent was distilled off from the boiling flask to about volume of 5mL, then, the extract was transferred into a 25mL volumetric flask, and filled to the mark with the methanol.

Both extraction methods also able to extract compound like resveratrol, piceid and emodin. SFE is more advantage than Soxhlet extraction because under optimized condition of the SFE is faster than 4h lasting Soxhlet extraction and SFE do not require large amount of solvent.

After the extracts was prepare, the prepared extracts were analysed using HPLC-UV method. The liquid chromatograph GBC LC 1445 consist of LC 1150 pump, LC 1650 auto sampler and LC 1210 UV-VIS detector was used. The separation was carried out using LiChrospher100, RP-18 column (125mmÃ-4mm, 5μm) that connect with LiChroCart guard column. The separation conditions were mobile phase water with 0.1% (v/v) phosphoric acid and mobile phase acetonitrile, the gradient elution used were 0 to 10min, 10% acetonitrile, 10 to 20min, 10 to 40% acetonitrile, 20 to 25min, 40 to 80% acetonitrile and 25 to 35min, 80 to 100% acetonitrile. The flow rate was set at 0.5mLmin−1; the temperature of separation was about 35 C. The UV detection was carried out at 306nm. Mobile phase components and injected extracts were filtered through 0.45μm membrane filters. 20μL of extract was injected to the chromatographic system. The individual compounds were identified by comparison of retention times. The content of the target compounds was determined using the calibration curve method. The linear range was evaluated. Then, limits of detection and quantification were established. The limit of detection (LOD) and limit of quantification (LOQ) were established for the individual compounds. The LODs were determined using lower concentrations of standards of 3:1 (S/N=3). LOQs were calculated from S/N of 10:1. Linear regression analysis using the least squares method was used to evaluate the calibration curve of each analyte as a function of concentration. The linear range together with LOD and LOQ values for piceid, resveratrol and emodin are summarized and listed in table 5. For all curves, correlation coefficients were higher than 0.9981. Typical chromatograms of extracts obtained from both supercritical fluid extraction and Soxhlet extraction are shown in figure 22 and figure 23 (Blanka Benova et al., 2009). Stilbenes based compounds piceid and resveratrol and anthraquinone derivative emodins were chosen as target compounds. The optimal extraction pressure (40MPa), temperature (100 C), time (45min) and modifier (acetonitrile) were established. At last the resveratrol extract vacuum-dried to remove the solvent and ground into fine resveratrol powder, which in turn is combined with fillers such as rice flour or hydroxypropylcellulose to manufacture pills and capsules (Becker et al., 2006).

Table 5: Characteristics of analyzed compounds with retention times, limits of detection and quantification of LOD and LOQ in μgL−1, equations of linear regression (y is peak area while x is concentration of appropriate compound) and appropriate correlation coefficients R2. (Blanka Benova et al., 2009)a

Compound

Retention time (min)

LOD (μg L−1)

LOQ (μg L−1)

Regression equation

R2

Piceid

21.6

21

69

y = 100,470x − 18,278

0.9983

Resveratrol

24.3

8

29

y = 191,266x − 79,322

0.9991

Emodin

30.4

52

174

y = 24,860x − 39,542

0.9981

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Figure 22: Chromatogram of target compounds standard solution (A) and typical chromatogram of supercritical fluid extraction (SFE) extract of Japanese knotweed (B). (1) Unidentified compounds (2) stilbene glycosides (astringin and resveratroloside) (3) emodin glycoside derivates (4) emodin derivate (physcion). Compounds labeled 1-4 are not target compounds in this study. (Blanka Benova et al., 2009).

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Figure 23: Typical chromatogram of Japanese knotweed after Soxhlet extraction. (1) stilbene glycosides (astringin and resveratroloside) (2) emodin glycoside derivates (3) emodin derivate (physcion).Compounds labeled 1-3 are not target compounds in this study. (Blanka Benova et al., 2009).

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