Austin Goewert, Chem 213 Synthetic #2 FFR Isolation of Piperine from Black Pepper Introduction Extraction from natural

products is an important practice in chemistry because it is the source of many medicinal and pharmaceutical compounds. It is estimated 60% of all drugs currently on the market were derived from natural products.1Along with having a direct effect on the body, natural sources are also an important inspiration for new drugs. Modern techniques can easily determine the chemical components of a natural product and determine their various effects on the body.2As many natural sources have yet to be explored, it is important to continue to study extraction from natural products and the medicinal effects of natural compounds. The extraction of piperine from black pepper is one such natural extraction. Black pepper itself has many important biological properties including analgesic effects and anti-cancer effects. To gain a better understanding of these effects, it is necessary to study the components which make up black pepper. GC-MS analysis shows black pepper is composed of lignans, alkaloids, flavonoids, and oils. One particularly active compound is the alkaloid piperine. Piperine helps vital chemicals from other food products reach the body by protecting against breakdown in the intestine and then protecting against oxidative damage in the bloodstream. Piperine also helps to reduce cancerous tumor growth by inhibiting pro-inflammatory cytokines which in turn stops communication between cancer cells.3 Various methods have been successfully used to extract piperine from black pepper. The most common method involves reflux of black pepper with ethanol followed by an overnight precipitation of piperine using 10% potassium hydroxide in ethanol. An alternative method
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involving reflux with dichloromethane also results in a good yield of piperine. These two methods are quite similar; the main difference is the time it takes to complete the procedure. The quicker method is dichloromethane, involving a 20 minute reflux and immediate precipitation of product using ether. The ethanol method is more time consuming, involving a 90 minute reflux and overnight precipitation.4Since both methods produce similar yields of piperine, neither method is necessarily superior. The ethanol method was chosen for this experiment. The purpose of this experiment is to isolate piperine from black pepper by reflux with ethanol and precipitation with 10% KOH in ethanol. The piperine will be purified by recrystallization using 3:2 acetone/hexanes. The identity of the product can then be determined using melting point, NMR, IR, and MS analyses. Experimental Piperine.Finely ground black pepper (12.5g) was refluxed with ethanol (50mL) for 90 minutes. Vacuum filtration was used to collect the filtrate, which was then evaporated. The residual oil was dissolved in 10% wt. KOH in ethanol (12.5mL). Water (100mL) was slowly added, forming a yellow precipitate. The solution was sealed and refrigerated overnight. The precipitate was collected by vacuum filtration and washed with cold ether (8mL). Recrystallization (3:2 acetone/hexanes) afforded piperine as flaky, golden crystals (0.272g, 2.17%), mp 127-132oC; 1H NMR (400MHz, CDCl3)7.3999-7.3742(m, 1H), 6.9806 (s, 1H), 6.9023-6.8782 (d, 1H), 6.78826.6934 (m, 3H), 6.4605-6.4240 (d, 1H), 5.9762 (s, 2H), 3.6377-3.5259 (d, 4H), 1.6868-1.2527 (m, 6H); IR (ATR)max(cm-1) 2919.27, 2851.79, 1631.23, 1580.25; HRMS (ESI) m/z 285.

Results and Discussion

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Piperine was extracted from black pepper by reflux with ethanol. The piperine was then precipitated in the refrigerator overnight using 10% KOH in ethanol and water. The solid product was purified by recrystallization with 3:2 acetone/hexanes. The product was further analyzed by mp, 1H NMR, IR, and MS analyses. Piperine makes up a small fraction of black pepper. In order to successfully extract piperine, all the lignans, flavonoids, and additional alkaloids must be removed. The isolation begins with 90 minutes of reflux with ethanol. During reflux, a pressure buildup resulted in some mixture lost out the top of the condenser column. Since piperine is soluble in ethanol, this reflux served to pull the piperine from the pepper grounds into solution. Many other components of black pepper, such as the lignans and flavonoids are less polar then piperine and do not move into the ethanol solution. Following reflux, these less polar compounds were removed by vacuum filtration. Next, the filtrate was evaporated, leaving a thick, dark oil. The precipitation of piperine was initiated by dissolving this oil in 10% KOH in ethanol and water. Piperine is slightly soluble in water, so the cream colored solution was refrigerated to further decreasepiperines solubility. After refrigeration, the solid was purified by recrystallization with 3:2 acetone/hexanes. The final product was flaky yellow crystals which is consistent with the literature for this experiment.4 The melting point of the product was 127-132oC. This is consistent with the literature value of 130-131 oC.5The mass spectrometry result also suggests the presence of piperine. According to MS (Figure 1), the molecular weight of the product is 285 atm. This result is consistent with the known molecular weight of piperine. Furthermore, this shows that the

product cannot be piperanine or piperettine, two other alkaloids found in black pepper, as they each have a molecular weight higher then 285atm. The product was analyzed further by IR (Figure 2). The carbonyl substituent is shown as a sharp peak at 1580.25cm-1. The carbon-carbon double bonds can be observed as another sharp peak at 1631.23cm-1. The other identifying bonds in piperine are the vinylic and aromatic hydrogens, which are present as moderate peaks at 2851.79cm-1 and 2919.27cm-1 respectively. This IR spectrum exactly matches the piperine spectrum published by the integrated spectral database system of organic compounds.6Since the other alkaloids have similar characteristic bonds however, the IR data alone is not conclusive. The final analysis performed was 400Hz 1H NMR (Figure 4). The ten hydrogens around the ring containing nitrogen absorb the furthest upfeild. The four hydrogens closest to the nitrogen can be observed as a doublet from 3.64-3.52ppm. The other six hydrogens on the ring absorb even further upfield; they are present as a multiplet around 1.6ppm. The two hydrogens attached to the 5 membered ringon the opposite end of the molecule can be seen as a singlet at 5.98ppm. This leaves 7 vinylic and aromatic hydrogens, which can be observed in a variety of splitting patterns from 6.42-7.40ppm. These vinylic and aromatic hydrogens prove that piperine was the alkaloid isolated. Differing from piperine, piperettine has two additional vinylic hydrogens, so its NMR would have a total of 9 hydrogens in the ~6.4-7.4ppm region. Piperanine on the other hand has two less vinylic hydrogens then piperine and thus its NMR would only have 5 hydrogens in the ~6.4-7.4ppm region along with an additional four hydrogens further upfeild. The presence of 7 vinylic and aromatic hydrogens shows the presence of piperine. The 400 MHz NMR obtained matches that published by the integrated spectral database system of organic compounds.6
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Since all four of these analyses match the literature values for piperine, it can be concluded that the identity of the product is piperine. Furthermore, MS and 1H NMR data exclude the possibility of other alkanoids, proving the product is pure. The percent recovery of piperine was 2.17%. This is only slightly lower then the typical results of 2.5-10%.4The majority of product loss can be attributed to the pressure buildup during reflux. This error was not serious however, as a fair yield of piperine was still obtained. Should this experiment be performed in the future, greater care should be taken to avoid pressure buildups during reflux. This can be accomplished by ensuring the reflux apparatus is open to the air. Despite this small loss of product, the isolation of piperine was a success. Using black pepper as a natural starting material, reflux with ethanol separated out many of the less polar components. Cooling overnight in 10% KOH in ethanol successfully precipitated the piperine. Recrystallization afforded a pure piperine product as shown by mp, MS, IR, and 1H NMR. References 1. Newman, D.J. J. Med. Chem. 2008, 51, 2589-2599. 2. Hong, F.J.; Xue, J.L.; Hong, Y.Z. EMBO Reports. 2009, 10, 194-200. 3. Meghwal, M.; Goswami, T.K. Open Access Sci. Reports. 2012, 2, 172-177. 4. Epstein, W.W.; Netz, D.F.; Seidel, J.L. J. Chem. Ed. 1993, 70, 598-599. 5. Coden, T. Tetrahedron Letters. 1986, 27, 603-6. 6. Integrated Spec. Data. System of Org. Comp.(Japan)