Bradley S. Winegardner, Chem 213W
Synthetic #1 FFR
Experiment 25: Isolation of Piperine from Black Pepper
Introduction
The use of natural products as medicine has been practiced for thousands of years. Natural products can be very diverse and yield the best medicinal results because they may produce fewer side effects. Natural products are used as the starting products by pharmaceutical companies when trying to create new, modernized medicines. Making medicine out of natural products has had a lasting effect on organic chemistry because it paved the path for modern, medicinal research on pharmaceuticals and for the development of new, synthetic medicines synthesized in a laboratory setting.
The purpose of this experiment was to isolate
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piperine (Figure 1, Report), an alkaloid, from ground black pepper and purify it through recrystallization. Alkaloids are nitrogen-containing and natural compounds that are usually basic in charge. Black pepper is a spice commonly consumed in the diet and can also be used as a preservative. With this product being so extensively engrained in everyday life, it is important to discover any medicinal qualities that black pepper may have. Piperine has been shown to activate digestive enzymes, pancreatic and gastro, as well as having the capabilities to neutralize free radicals and reactive oxygen species, which can lead to cellular-oxidative stress. The chemical components found in piperine are amides, piperidines, and pyrorolidines. Piperine gives black pepper its’ pungency because it is a lachrymator and can cause the eyes to tear up or the nose to run. The specific isolation technique for this experiment was reflux.
Reflux is an isolation technique that heats a reaction mixture to produce a distillate that travels up a reaction vessel and is cooled using a reflux condenser (Figure 2, Report) and cold water, which will condense the distillate back into the reaction vessel. The piperine was in this liquid portion of the reaction mixture. The condensate was filtered away from the black pepper and reduced to a yellow, powder residue under a stream of nitrogen and a hot water bath. The residue was dissolved in potassium hydroxide and ethanol and then precipitated with the addition of DI water. The precipitant was filtered away and this concluded the isolation of the crude piperine product. The purification of piperine was done by recrystallizing the crude product in a hexanes/acetone solvent mixture and the crystals filtered away from …show more content…
solution. The purified piperine product was analyzed using melting point characterization, 1H NMR, IR, and MS. Experimental Piperine. Black Pepper (12.5 g) was ground and refluxed with ethanol (50 mL) for 90 minutes. The reaction was stopped and cooled. The reaction contents were filtered off and the black pepper thrown away. The resulting liquid was reduced to a yellow powder and the product dissolved in potassium hydroxide (10%, 1.6 g KOH in 20 mL ethanol, 0.029 mmol)3 and ethanol (12.5 mL). DI water was added to resulting solution (100 mL) and the reaction chilled for 2 days. The precipitant (1.048 g, 8.38%) was collected and dried for 1 hour, then dissolved in hexanes/acetone solvent mixture (1:1 ratio, 7 mL). The solution was brought to room temperature for 15 minutes and chilled for another 30 minutes. The resulting crystals were vacuum filtered to yield a coarse, yellow solid (0.458 g, 3.66%). Melting point characterization (ºC) 108.2-142.6; 1H NMR (60 MHz, CDCl3): δ (ppm) 7.293 (t, 1H & s, 1H) 6.992 (d, 1H) 6.813 (d, 1H & d, 1H & t, 1H & d, 1H) 6.002 (s, 2H) 3.588 (t, 4H) 1.643 (q, 4H and q, 2H); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.3742 (t, 1H) 7.2698 (s, 1H) 6.9844 (d, 1H) 6.9023 (d, 1H) 6.8822 (d, 1H) 6.7882 (t, 1H) 6.4605 (d, 1H) 3.5644 (t, 4H) 1.5876 (q, 4H & q, 2H); IR (ATR) υmax (cm-1) 2922.5-2853.6, 1629.0, 1246.6; MS (Piperine) m/z 285. Results and Discussion In this experiment, the piperine product was isolated via reflux condensation from natural black pepper. The crude product was then purified using recrystallization and analyzed by melting point characterization, 1H NMR (60 MHz and 400 MHz), IR, and MS. The isolation and purification of piperine from black pepper was done because it is a natural product with naturally occurring medicinal properties.
Piperine has been shown in studies to reduce the risk of cancer by neutralizing free radicals within the body as well as eliminate reactive oxygen species. Both free radicals and ROS can cause cellular oxidative stress and may lead to some cancers as well as early onset of aging, and certain genetic mutations. Piperine also has the ability to stimulate the pain-sensing nerves and this gives pepper its’ pungency. Piperine also aids in digestion by triggering pancreatic and gastro digestive enzymes that will chemically break down the food that we have consumed. Piperine is such an important part of the human diet (in the form of black pepper) and because it is a natural product, there are fewer side effects associated with piperine as a medicine. Natural products as medicine are so important because they allow pharmaceutical companies to research their effects without so many associated risks and can lead to the development of synthetic, modern pharmaceuticals based on the characterization of the natural products. The research of natural, medicinal products leads to many medical
advancements. The isolation of piperine from black pepper was accomplished by refluxing a ground black pepper and ethanol solution in a reflux condenser. The condensation of this reaction contained the crude piperine product and was filtered away from the black pepper using vacuum filtration. The condensate was reduced to a yellow; powder residue under a stream of nitrogen and over a hot water bath and the residue dissolved in 10% potassium hydroxide (1.6 g KOH in 20 mL ethanol)3 and ethanol. DI water was added to the solution mixture to precipitate the crude piperine product and the mixture was chilled for two days to encourage precipitation. The crude product was filtered away from the solution and dried for one hour. This concluded the isolation of piperine from black pepper. The purification of the crude piperine product was accomplished via recrystallization. The crude product was suspended in a hexanes/acetone solvent mixture because the piperine product is non-polar due to having very little hydrogen bonding capability, a large carbon-hydrogen to electronegative atom ratio and no dipole1. With the hexanes/acetone being a non-polar solvent it makes it a perfect recrystallization solvent due to the similarity in the polarities of the piperine and the solvent mixture. The solution was left at room temperature for 15 minutes and then chilled on an ice/salt water bath for 30 minutes. The crystals were then extracted via vacuum filtration and this concluded the purification of piperine. Recrystallization was a viable source of purification because of the vast difference in polarity of the ethanol and piperine. The crude product was obtained as a yellow residue (1.048 g piperine) in 8.38% recovery from the black pepper. This compared well to the typical range of percent recoveries for this experiment in Chem 213W from previous semesters (2.5-10%)1,4. The percent recovery for the recrystallization, 43.7%, was respectable in the fact that it was close to 50% and that there was probably more ethanol that desired remaining in the piperine residue. The percent recovery for the purified piperine from black pepper was 3.66% and although very low still fell within the typical students’ ranges for Chem 213W. A plausible explanation for such low yields would be that piperine only exists as 10% of the weight of black pepper and it is impossible to extract all of even this small amount of piperine because in order to do so the black pepper would have no form to its structure (i.e. ground so fine it would no longer be a powder). The purified product was characterized by IR, MS, and 1H NMR (60 MHz and 400 MHz). The IR analysis (Figure 1, Supplemental Information) supports the formation of piperine as a product. However, this spectrum does not support the purification of piperine from the ethanol solution. The aromatic ring structure in piperine shows up between 2922.5-2853.6 cm-1, the carbonyl shows up at 1629.0 cm-1 and the C-O bond in the five membered ring shows up at 1246.6 cm-1. Although the C-O bond shows up in the fingerprint region of the IR analysis, it was predicted that the C-O bond would show up at 1270-1200 cm-1 and can be concluded that this is representative of the C-O bond. The impurity in the sample showed up at 3500-3000 cm-1 in the form of a weak, broad peak indicating a hydroxyl (-OH) group. This is indicative that although the piperine was formed, there was also some ethanol contamination in the sample analyzed. The 1H NMR (Figure 2 and 3, Supplemental Information) confirms the formation of piperine revealing a triplet at 7.3742 ppm representing an alkene hydrogen, a singlet at 7.2698 ppm representing a benzylic hydrogen on the aromatic ring structure, and 5 more peaks at 6.4-7.6 ppm representing the other benzylic and alkene hydrogens. There was a singlet at 5.9762 ppm representing the –CH2 hydrogens on the five membered ring structure and a triplet at 3.5644 ppm representing the four hydrogens located on the six membered, nitrogen containing ring. Two quintets showed up at 1.5876 ppm clarifying the other six hydrogens located on the same six membered ring, nitrogen containing structure. There were no impurities found in the 1H NMR spectra and this could be because these samples were diluted in CDCl3 whereas the IR sample was the dry, purified product. The MS characterization (Figure 4, Supplemental Information) was the final analysis and showed that the mass of the sample ran was 285 grams by molecular weight and this was predicted (piperine, MW: 285.34)2. This analysis showed that there was a six membered, nitrogen containing ring at the end of the product (-84 g) as was predicted, followed by a carbonyl compound (-28 g) also as predicted. This indicated that the compound submitted for analysis was indeed piperine. In conclusion, the reflux condensation of black pepper and ethanol isolates a piperine product that was purified via recrystallization is a hexanes/acetone solvent mixture. The condensation reaction was successful at forming a crude product, piperine, at a viable recovery (8.38%) and the recrystallization of piperine was successful at giving a good yield (43.7% recovery) and being a quick, inexpensive purification method but not successful at producing a pure product as indicative of the distinguishing ethanol peak on the IR spectrum. In the future, the purification of piperine should be carried out in a column using flash chromatography. This method is more expensive and more time consuming; however, it would yield a more pure product. References 1. Rummel, S.A., Beiswenger, K.M., ED. Introductory Organic Chemistry Laboratory; Department of Chemistry: Penn State, Hayden-McNeil Publishing, Plymouth, MI, 2016. 2. Piperine; MSDS No. P49007, Sigma Aldrich, www.sigmaaldrich.com (accessed 3/23/16) 3. Potassium Hydroxide; MSDS No. P5958, Sigma Aldrich, www.sigmaaldrich.com (accessed 3/23/16) 4. Epstein, W. W.; Netz, D. F.; Seidel, J. L. J. Chem. Ed. 1993, 70, 598-599 5. Vasavirama, K.; Upender, M. Int. J. Pharm. Pharm. Sci. 2014, Vol 6, Issue 4, 34-38. 6. Hu, L.; Hao, C.; Fan, R.; Wu, B.; Tan, L.; We, H.. J.Pone. 2015, 10, 1371. 7. Raman, Girija; Gaikar, V. G.; Ind. Eng. Chem. Res.2002, 41, 2966-2976.
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A condenser and heat reflux was used to prevent reagents from escaping. Then the solid product was vacuum filtered. The product was recrystallized to purify it and the unknown
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Nagaland of India contains one of the world’s hottest chilies – the Naga King Chili (Bhut Jolokia). The author of the article, Mary Roach, arrived to Nagaland to experience the Hornbill Festival, in particular, to watch the Naga King Chili-Eating Competition. She aimed to find out and understand the pleasure and pain the Capsicum genus brings to the people who consume it. The Naga King Chili Pepper varies in heat, but can score extremely high on the Scoville heat unit. The contestants of the chili-eating contest began to suffer from the effect of the capsaicin in their digestive tracts. Some experienced hot flashes and unbearable heat from within, others, like the winner of the contest, vomited at the end of the competition. Some competitors were only able to eat a few of the Naga King Chili, however, others ate up to fourteen chilies. Tolerance for capsaicin, according to the article, is largely built on practice. Only a small part is due to genetics. Capsaicin damages pain receptors over time, so if someone continuously eats hot chili peppers, the pain receptors in their nerves will slowly degenerate and become less sensitive. Mary Roach was able to sample the Naga King Chili in small doses, which brings out the wonderful flavors that would otherwise be overwhelmed by the heat of the capsaicin.
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The major use for herbal medicines is for the promotion of health and for therapy for chronic conditions, instead of being used for life-threatening conditions; except in the event of say advanced cancer or new infectious diseases when conventional medicine practices no longer are working an individual may use traditional remedies. While traditional medicines are often mistaken that because they are natural that they are safe, non-toxic, which is not always the case. In cases when an individual is taking herbs with prescription drugs, over-the-counter medications, or other herbs that may cause some adverse side affects. As a flourishing commercial enterprise, it doesn’t matter why an individual uses traditional medicines, it provides important health care services for individuals that have access physically or finantually to allopathic
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The 20th Century gave birth to many new advancements in medicine. During this time, the average lifespan was lengthened, due to progress in medical discoveries. However, with old age came an increase in diseases associated with aging, including cancer and heart disease, which led to increased study’s on how to prevent or cure such illnesses. By the beginning of the 20th century, a new term had emerged, “pharmacology”, which is a laboratory science concerning the study of natural substances and remedies. Researchers put plant drugs under systematic chemical analysis and later learned how to synthesize the drugs. By the turn of the 20th century, new medicines were being marketed and sold in pharmacy’s. Bayer, a company in Germany, made a synthesized version of acetylsalicylic which is what we know today as Aspirin.[1]
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