EXTRACIA CAPSAICINEI DIN ARDEIUL IUTE

GeneralitiCapsaicina se extrage n principal din ardeiul iute. Parte experimentalCapsaicina se extrage din ardeiul iute folosind solveni organici (alcool acetona, acetonitril, hexan) i metode diferite de extracie.

1. Extracia capsaicinei prin macerarea ardeiului iute n alcool.Ardeiul se mrunete i se adaug ntr-un borcan nchis ermetic. Peste ardei se introduce alcool etilic concentrat (sau vodca) pn ce planta este complet imersat n alcool. Se menine, la soare cel puin 48 ore, dup care se filtreaz se concentreaz prin evaporarea parial a alcoolului i se introduce ntr-un recipient nchis ermetic. Se adaug puin amestec la o crem de corp i se obine o crem mpotriva durerilor musculare.

2. Extracia capsaicinei cu acetonPatru kilograme ardei iute se mrunesc n bender fr a aduga nici un fel de lichid. Amestecul obinut este uscat prin liofilizare i meninut la -30C. Se cntresc 100 g material uscat i se extrag, timp de 2 h cu 1,5 l aceton, la rece. Dup 2 h se filtreaz lichidul. Procesul se repet pn soluia extras este incolor (cca 3-4 ori). Final materialul extras rmne sub form de pulbere fin de culoare uor glbuie. Soluia de aceton (de culoare verde nchis datorit extraciei clorofilei) sunt amestecate i concentrate la rotavaporizator. Amestecul concentrat rmas (ce conine oleorini) este diluat cu ap deionizat luat de 4 ori volumul soluiei concentrate, apoi adus la pH=11 cu sol. 0,5N KOH. Amestecul se dilueaz n continuare cu 1,5 volume ap deionizat, dup care se aduce la pH=7,5 cu soluie HCl i se extrage de 3 ori cu eter etilic ntr-o plnie de separare. Eterul se distil la vid i blaz se recolteaz un ulei vscos de culoare brun-verzui.

3. Extracia capsaicinei prin metoda SoxhletArdeiul se taie n sferturi, se separ manual seminele (acestea au cel mai mare coninut n capsaicin) i se taie ct mai fin sau se mrunesc la bender. Se usuc materialul vegetal n etuv la temperaturi sub 100C.Se monteaz balonul pe o plit de nclzire sau pe o baie de ap i se adaug 100 ml etanol 95%. Se monteaz instalaia Soxhlet i se ncepe nclzirea. Refluxarea se continu pn cnd etanolul ce se adun n piesa intermediar este incolor. Extractul se concentreaz la rotavaporizator sau distilare la vin a etanolului. Ametecul concentrat se menine peste noapte ntr-un flacon nchis ermetic.

Atenie!!!!Capsaicina pur provoac arsuri ale pielii n cantiti foarte mici. La fel i oleorina extras din ardei. Cnd lucrai folosii echipamentul complet de protecie: ochelari, mnui, halat, i, dac e posibil, i masc mpotriva prafului.

1. If available, use a rotary evaporator with a bump-trap to remove solvent.2. Set up a distillation apparatus, evaporate solvent under reduced pressure carefully to prevent bumping3. Allow concentrate to stand overnight in a sealed vial once cool. Oil will form separate layer.4. Use pasteur pipette to remove bottom oil layer (red-brown).Characterization of Scoville Rating: A panel of 5 volunteers tasted 1:10 serial dilutions of the original concentrate in 5% sucrose. Spicy taste/sensation was noted at the 1:100 dilution by 4/5, and again at 1:10 with 4/5 (different individual as the negative result individual) Concentrate was tasted after concluded to be safe enough to try volunteers noted that the concentrate was comparable to tobasco sauce.Future improvements: Avoid the vacuum distillation process, as some capsaicinoids could have been lost at this step Obtain better peppers, dry fully, and grind up well (this step was bypassed in lieu of filming the video quickly) Perform TLC to determine composition of dilute extract and concentrate.

AbstractA new method based on pressurized liquid extraction followed by LC-MS/MS analysis has been developed for the identification and quantification of three capsaicinoids (capsaicin, dihydrocapsaicin, and nordihydrocapsaicin) in extracts of Capsicum annuum. For the recovery of three capsaicinoids, the efficiency levels of ultrasonic-assisted extraction, microwave-assisted extraction, Soxhlet extraction, and pressurized liquid extraction were compared under different conditions. Pressurized liquid extraction resulted in higher yields. Pressurized liquid extractions were performed using methanol; temperature was set at 100C and pressure at 1500 psi. LC analysis was performed on a Waters XBridge C18 column (150 2.1 mm, id 3.5 m) eluted by a mobile phase of 0.1% formic acid and ACN. Data acquisition was carried out in multiple reaction monitoring transitions mode, monitoring two-reaction monitoring transitions to ensure an accurate identification of target compounds in the samples. The proposed method is rapid, simple, and could be utilized for the routine analysis of three capsaicinoids in C. annuum samples.

A simple method for the analysis of capsaicin and dihydrocapsaicin in peppers by ultrasound assisted extraction (USAE) followed by gas chromatographymass spectrometry (GC-MS) has been developed. USAE conditions were optimized by experimental design in order to maximize analyte extraction. A full factorial design involving extraction variables such as solvent (ethanol and water), extraction time (525min), extraction temperature (2550 C), sample amount (0.250.5g), and ultrasound amplitude (4080%) was applied. The most significant conditions for capsaicinoid extraction by USAE were solvent type, extraction time, and sample amount. The obtained results were compared with other extraction methods: the official Soxhlet method and a previously reported solid phase microextraction method. Results showed that the extraction efficiency with the application of USAE (98%) was as good as that obtained with Soxhlet and the precision of recovery was less than 5%; in addition, the extraction time was decreased from 5h to 25min. The GC-MS analytical method was linear in the range 10100g/mL for capsaicin and dihydrocpsaicin with correlation coefficient r=0.998 and peak area variability of1% for both capsaicinoids. The method was applied to the analysis of 11 varieties of hot peppers cultivated in Mxico. A large concentration range for capsaicin (1016800g/g) and dihydrocapsaicin (1102736g/g) was found in these pepper samples.

Chili peppers are the fruits of the genus Capsicum. There are several chili pepper varieties and they havebeen domesticated for years. Chili peppers are used to season foods in many cultures and they havebecome a staple in the diets of Italians, Spaniards, Hungarians, Indians, Chinese, Indonesians, andAfricans.The chemicals that cause the sensation of heat when you eat a pepper are called capsaicinoids. Capsaicinbeing the main capsaicinoid found in chili peppers. This chemical is so hot that just one drop mixed with10,000 drops of water will cause the tongue to blister.When you eat a pepper the capsaicin irritates the lining of your mouth causing that familiar burningsensation. Your brain responds to this stimulus by producing endorphins, your bodys own natural painreliever. The production of endorphins in response to eating chili peppers may explain why they can beaddicting.The capsaicin found in peppers also has medicinal uses. It relaxes the arteries (which lowers bloodpressure) and can be used in creams as a topical pain reliever. Currently, capsaicin and capsaicinderivatives are used to treat arthritis, shingles, diabetic neuropathy, neuralgia, and as a pain reducerfollowing surgery. Some studies have even shown that consumption of capsaicin-containing peppersreduces the amounts of clotting protein in you blood, thereby reducing a persons risk of having a stroke.The hotness of a chili pepper is measured in Scoville units. These units were invented byWilburScoville, a pharmacist, in 1912. To determine the hotness using Scoville units, a panel of 5 tasterssample extracts of the peppers that are diluted in sugar water. The dilution that can be detected by 3 of the5 tasters is considered the heat level of the pepper.The ratio of the extract dilution is the Scoville unit. For example, the jalapeno is usually detected by 3 of5 tasters when diluted at a ratio of 1/2500 to 1/5000; the Scoville unit for the jalapeno is 2500 to 5000.Just to compare, the habanero pepper can have 100,000 to 300,000 Scoville units.In this lab, you will be using a variation of Scovilles test to determine the hotness of several types ofcapsicum peppers.Materialsglass mortar & pestle 5 2-dram vials with caps graduated pipettes latex gloves5% sucrose solution 95% ethanol electronic balanceweighing boat knife various chili peppers

Procedure1. Get into a group of 5 an obtain the necessary materials. Each person in your group should obtaina different type of pepper.2. Label the dram vials 1:10, 1: 100, 1: 1,000, 1: 10,000, 1:100,000.3. Fill each dram vial with 4.5 mL of 5% sucrose solution.(If you are unsure about how to read the pipette, please ask.)4. Place the weighing boat on the electronic balance. Zero the balance by pushing the zero button.5. Cut off a small piece of the pepper and place it in the weighing boat. You want a 5 grams sample ofpepper. Cut off or add more pepper until the balance reads 5 grams (re-zero the balance ifnecessary).6. Place the pepper sample in the glass mortar and add 5mL of 95% ethanol. Use the pestle to grindup the pepper in the ethanol.7. Use a clean pipette to transfer 0.5mL of the extract into the vial marked 1:10. Cap and shakethe vial.8. Use a clean pipette to transfer 0.5mL of the extract in the 1:10 vial into the vial marked 1:100.Cap and shake the vial.9. Use a clean pipette to transfer 0.5mL of the extract in the 1:100 vial into the vial marked1:1,000. Cap and shake the vial.10. Use a clean pipette to transfer 0.5mL of the extract in the 1:1,000 vial into the vial marked1:10,000. Cap and shake the vial.11. Use a clean pipette to transfer 0.5mL of the extract in the 1:10,000 vial into the vial marked1:100,000. Cap and shake the vial.12. Place a clean pipette in the 1:100,000 vial.13. Perform the taste test with the other four members of your group. Using the clean pipette in the1:100,000 vial to place a drop of the solution on each persons coffee stirrer. All members shouldplace the drop on their tongues and determine if they can detect the capsaicin.14. Move the same pipette (you do not need a new one for each vial) to the next vial (1:10,000) andrepeat the taste test. Continue until at least three people in your group can detect the capsaicin inthe solution, then mark the appropriate box in the data table.15. Eat an unsalted cracker and drink some water.16. Repeat steps 12-15 until all 5 peppers have been tested.17. Record the Scoville units in the data table. Remember that if capsaicin is detected at the 1:10,000dilution, then the Scoville unit for that pepper is 10,000.18. Wash and dry all of the materials and place them back in the proper place. Your box of materialsshould have the mortar & pestle, 10 dram vials, and 15 pipettes in it before you leave the room.

AOAC Official Method 995.03Capsaicinoids in Capsicums and Their ExtractivesLiquid Chromatographic MethodFirst Action 1995[Applicable for determination of 750-650000 Scoville Heat Units (SHU) ofcapsaicinoids in ground and crushed red pepper, chili pepper, ground cayennepepper, ground jalapeno pepper, and red pepper oleoresins. Not applicable to chilipowders or products containing oregano or thyme.](Caution: See Appendix B, safety notes for the safe handling of organic solvents andspecial chemical hazards-acetone, acetonitrile, and ethanol. See Material SafetyData Sheets, or equivalent, for each reagent. N-Vanillyi-nnonanamide is an extremeirritant; do not inhale. Dispose of waste solvents in an appropriate mannercompatible with applicable environmental rules and regulations.)Method Performance:See Table 995.03 for method performance data.A. PrincipleTest sample is extracted in warm ethanol using reflux condenser. Extract is filteredand injected into liquid chromatograph equipped with UV or fluorescence detector.B. Apparatus (a) Liquid chromatograph (LC).--Equipped with I V integrator, 20 gLsample injector, and with UV detector set at 280 run. wavelength or fluorometerwith excitation 280 nm and emission 325 nm. Operating conditions: temperature,ambient (20-25'C); flow rate, 1.5 mL/min., isocratic; relative retention times: Nvanillyl-n-nonanamide, 1.00; nordihydrocapsaicin, 0.90; capsaicin, 1.00;dihydrocapsaicin, 1.58. See Figure 995.03 for baseline separation of majorcapsaicinoids.(b) LC column.--Stainless, C18, 150 x 4.6 mm id, packed with 5 micrometer particlesize. Use guard column, if desired.(c) Reflux condenser.(d) Syringe filter.- 0.45 micrometer.(e) C18 solid-phase extraction cartridge.C. Reagents(a) Ethanol-95% or denatured, suitable for chromatography(b) Acetone.-ACS grade.(c) LC mobile phase@Acetonitrile-water. Use LC grade solvents, or equivalent. Mix400 mL acetonitrile with 600 mL H20 containing 1% acetic acid (v/v). De-gas withhelium or by other suitable technique.(d) N-Vanillyl-n-nonanamide standard solutions. -N-Vanillyln-nonanamide standard,99%, is available as synthetic capsaicin from Penta International Corp., 50 OknerPkwy, Livingston, NJ 07039. Keep solutions out of direct sunlight. (1) Standardsolution A.-0. 15 mg/mL. Accurately weigh 75 mg N-vanillyl-n-nonanamide andtransfer it into 500 mL volumetric flask. Dilute to volume with ethanol, and mix.Use standard solution A for analyzing all peppers except chili pepper.(2) Standardsolution B.-0.015 mglmL. Transfer 10 mL standard solution A into I 00 mLvolumetric flask, dilute to volume with ethanol, and mix. Use standard solution Bwhen analyzing chili peppers.D. Extraction(a) Ground or crushed peppers.-Accurately weigh ca 25 g pepper into 500 ITILboiling flask. Place 200 mL ethanol into same flask, add several glass beads, andattach flask to reflux condenser. Gently reflux test sample 5 h and then allow tocool. Filter 1-4 mL sample through 0.45 gm syringe filter into small glass vial. Usefor LC analysis.(b) Red pepper oleoresins.-Accurately weigh 1-2 g oleoresin into 50 mL volumetricflask. Increase weight of sample, if total capsaicinoid concentration is < 1 %.Note: Do not allow any oleoresin to coat sides of flask.Add 5 mL acetone, C(b), to flask and swirl contents of flask until test sample iscompletely dispersed (no oleoresin can coat bottom of flask when turning flask neckat 45' angle). Add five 3-5 mL portions ethanol, swirling flask during each addition.Dilute contents of flask to volume with ethanol and mix well.