HERMIN HARDYANTI UTAMI (111 304 0202)

CHEMISTRY DEPARTMENT MATHEMATIC AND SCIENCE FACULTY

STATE UNIVERSITY OF MAKASSAR 2012

APPROVAL SHEET Complete report of organic chemistry II with the tittle Piperine Isolation from Pepper and Chemical Properties of Piperine was created by : Name Reg. Number Group Class accepted. Makassar, December 2012 Assistant Coordinator Fandi Ahmad, S.Pd Known by Responsibility Lecturer Drs. Hj. Ramdani, M.Si. NIP : 19630221 198803 2 001 Assistant Yanti Madong, S.Si. : Hermin Hardyanti Utami : 1113040202 : VII (seventh) : ICP Chemistry B

after corrected and consulted by assistant and assistant coordinator, so it was

A. Title of experiment Piperine Isolation from Pepper and Chemical Properties of Piperine B. Purposes of experiment At the last experiment, student can be understand about :
1. Isolation principle of organic compound from nature material, especially

alkaloid group.
2. Isolation technique in nature material, continue extraction, separating, and

it purification.
3. Knowing chemicals properties of piperine based on the structure and it

degradation result. C. Background A natural product is a compound synthesized by a plant or an animal. Alkalords are natural products that contain one or more nitrogen hetero atoms and are found in leaves, bark, roots or seeds of plants. Examples include caffeine (found in tea leaves, coffee beans, and cola nuts) and nicotine (found in tobacco leaves). Morphine is an alkaloid obtained from opium, the juice derived from a species of poppy. Morfphine is so times stronger than aspirin as an analgesic but it is addictive and suppresses respiration. Heroin is a syntetic compound that is made by a cetylating morphine. (Bruice, P.Y. 2003:884). With identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amount amino acid are linked together. Much peptide sequencing is now done by mass spectrometry, using either electropspay ionization (ESI) or matrix-assisted laser desoroption ionization (MALDI) linked to a time of flight (TOF) mass analyzer as described. Also in common use is a chemical method of peptide seqencing by called the Edman degradation. Edman degradation involves treatment of a peptide with phenil isothiocyanale (PLTC), C6H5-N = C = S, followed by treatmen with trifluoroastic acid. The first step splits tne N-terminal residue from the peptide chain, yielding an anilinotr zlinone (ATZ) derivative with shortened peptide. Futher acid catalyzed rearrangement of the ATZ derivative with aqueous acid converts it into a

phenylthrohydantion (PTH), which is derivative with aquous acid converts it into by chromatographicaly by comparasion if its solution time with the known elution times of PTH derivaties of the 20 common amino acids. The chain-shortened peptide is then automatically resubmitted to anther round of Edman degradation (Mc Murry, John. 2008 : 1031). Protein metabolism. Catabolism of proteins: transamination 1. The pathway to amino acid catabolism a. The amino group is removed by transamination b. What remains is converted a compound that enters the citric acid cycle. 2. Transamination a. The NH2 group of an amino acid adds to the aldehyde group of pyridoxal phospate to form an imine. b. The imine tautomerizes to different imine. c. The second imine is hydrolyzed to give an derivative of pyridoxal phosphate. d. The pyridoxal derivate transfers its amino group to regenerate pyridoval phasphate and form glutamate. 3. Deamination The glutamate from transamination undergoes oxidative deamination to yield ammonium ion and -ketoglutarate (McMurry, Susan. 2008:796). Spectrophotometric metods, involving UU absorbance measurements at the pipenrine absorption maximum near 343 nm measure predominantly piperine but include some contribution from piperyline, piperettine and any other components having substantial absorption at this wave lenght. Piperine is extracted from ground pepper by refluxing 3 hour with ethanol (96% v/v) whilst mogenized oleoresins are simply dissolve solvent (Wood, A.B. 988:55). Piperine [(1-5-(1,3) benzodioxol -5 yl.) . 1 -0 x 0 2, 4 pentadieny] piperidine) an alkalord responsible for the pungency of black that pepper and long pepper. Systemic pharmacological studies on the piperine have revealed that this compound elicited diverse was pharmacological activities, analgesic, anty-pyretic, antrinflammatory, anti convulsant and CNS depressant activities. Piperine was -ketoglutarate, to keto acid and an amino

isolated from piper nigium Linn. (Piperaceae) and identified by TLC. Piperine was fabricated into alginate beads by using sodium alignate. The main aim of this study was to demonstrate that the sutained release of piperie from alginate beads by invitro evalution. The drug release studies were showed that the alginate beads sustained the release of drug with % drug released in hours (Madhavi, B.B. 2009:156). D. Chemical and Equipment 1. Chemical a. Pepper sample b. Aquadest (H2O) c. Ethanol (C2H5OH) solution d. Alcoholist potassium hydroxide (KOH alcoholist) solution e. Potassium Permanganate (KMnO4) solution f. Concentrated hydrochloric acid (HCl) solution g. Aqueous hydrochloric acid (HCl) soution h. Aqueous sodium hydroxy (NaOH) solution i. Molisch reagent j. Benedict reagent k. Aluminium foil l. Matches m. Litmus paper n. Universal indicator o. Filter paper p. Whatmann filter paper. 2. Equipment a. Soxclet equipment b. Analytical scale c. Buchner funnel d. Test tube e. Test tube rack f. Stir bar (1 set) (1 piece) (1 piece) (4 pieces) (1 piece ) (2 pieces)

g. Dropping pipette h. Beaker glass 50 ml and 250 ml i. Funnel j. Graduated cylinder 10 ml, 25 ml and 100 ml k. Bunsen burner l. Triangle m. Asbestoz Gauze n. Spray flask o. Water bath p. Round flask 250 ml q. Soft cloth and rough cloth E. Work Procedure 1. Piperine Isolation

(5 pieces) (2 pieces) (2 pieces) (1 piece) (1 piece) (1 piece) (1 piece) (1 piece) (2 pieces) (2 pieces) (1 piece)

a. 20 grams of pepper sample was balanced on analytical scale and was filled into soxchlet that has covered by filter paper. b. It was added by ethanol with 3 times of circulation. c. It was soxcheletd by 15 times circulation. d. It was let stand and added by 60 ml alcoholist KOH. e. It was heated on water bath until concentrated and stirred. f. It was filtered by funnel and divided into 3 part (for piperine isolation, degradation, and it reaction). g. It was decreased the volume and let stand on the temperature until 1 day. h. It was filtered on Buchner funnel and the residue was took. 2. Piperine degradation a. 1/3 part of isolation result was decreased the volume by reflux it. b. It was added by HCl 10 M and stirred until dissolve and forming yellow precipitate. c. It was let stand on the temperature until 1 day. d. It was filtered on Buchner funnel and the residue was took. 3. Piperine reaction a. 3 pieces of test tube was filled by 2 ml of piprine isolation result.

b. It was added by HCl and reacted with : 1)Test tube 1st was reacted by KMnO4 2)Test tube 2nd was reacted by benedict reagent 3)Test tube 3rd was reacted by molisch reagent. c. Each was neutralized the pH and was checked by litmus paper. F.Observation result No 1. 2. 3. Activity Isolation of Piperine Balanced dust paper on the analitycal scale Covered with filter paper, filled to soxlet and added ethanol The mixture soxlet 1st circulation 2nd circulation 3rd circulation 4th circulation 5 circulation 6th circulation 7th circulation 8th circulation 9th circulation 10 circulation 11th circulation 12th circulation 13th circulation 14th circulation 15th circulation 4. 5. 6. Soxlet mixture heat 10 minutes Added 60 ml KOH alkoholis Stir and filtered
th th

Result 25.0124 g 3 times circulation of soxlet

11 minutes 7 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes 6 minutes Yellow solution Yellow solution Yellow solution

7.

Devided the solution

a. 6 ml reaction b. 59 ml for vapor (isolation) c. 59 ml for reflux (degradation)

8. 9. 10. 1. 2. 3. 4.

Vaporated until saturated (b) Let it stand 1 day Filtered it with ethanol Degradation (c) 59 ml solution reflux Vapored it until 10 minutes Added 10 M 10 ml HCl and 7 ml H2O Filtered with ethanol Piperin reaaction

Brown solution Brown solution Not crystall Dark yellow solution Yellownish brown solution Yellownish brown Not crystall

1.

2 ml piperin+HCl dilute Added NaOH 5 drops KMnO4

Acid solution Unknown pH 2 layers : 1st :yellow 2nd: turbid (negative)

2 ml piperin+HCl dilute Added NaOH 5 drops Benedict

Acid solution Unknown pH Greenish brown (negative) Acid solution Unknown pH Yellow solution (negative)

2 ml piperin+HCl dilute Added NaOH 5 drops Molish

G. Data Analyzed 1. Isolation of piperine Known. Weight of sample : 25.0124 g Mr of Pyperine (C17H19O3N) : 285 g/mol Mr of pyperidine (C5H10NH) : 85 g/mol Question=.?

Mass of theory = = 2. Degradation of Piperin Dik : Weight of sample : 25.0124 g Weight molecule of pyperine (C17H19O3N) : 285 g/mol Weight molecule of piperidine hydrokloride (C5H10NHCl): 120,5 gram/mol Weight of theory = = = 0,4228 H. Discussion 1. Isolation of piperine Isolation is seperating process organic compound from nature. In this experiment would be isolated is pepper that have done smoothly and technique that we used is continue extraction(soxletation), separation and purity from another compound that contain in black pepper. The principle soxletation is pulling chemical compound that done by the dust put on the filtering paper, the liquid heated in round flask until vapor and condetation by condensor ball become liquid molecule that drops until in the syfon surface, all of liquid will be down and back to theflask occured capiler pipes until became circulation. In this experiment the sample is black pepper that want to get the piperine. Piperine is the alkaloid responsible for the pungency of black pepper and long pepper, along with chavicine (an isomer of piperine). Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms. At this experiment, solvent that used is etanol. Etanol as solvent to solute all of compound that want in black pepper. Etanol used because has same polarity is polar until ethanol can solute as the prinsiple like dissolve like. Ethanol used because volatil properties where in hot condition easy soluble piperin and in cold condition easy to formed crystall. Then we do extraction continue until 15 times = 0.2982

circulation. At circulation process used boiling stone in flask solvent that purpose is to keep the pressure and temperature of solution for stable and not occured explosion. Process that happen belong circulation is ethanol solvent heated in round flask until vapor and colded used condensor, until fall become liquid to sample (black pepper) to soluble active element in sample black pepper and if liquid solvent have done reached the bottom of syfon then all of liquid from ethanol will be solute that have in syfon surface will be out occured small pipes to round flask in this process will occured continue and occured soxletation process, that process we call the principle of continue extraction. From soxletation process will be produced 124 ml extraction and in soxletation process occurs 15 circulations produced transparant yellow extract. After that, extract will dried belong 10 minutes until the solution concentration. It is because to vapor ethanol that contain in solution. Then added KOH alkoholis 10%. The function is to bond piperine from solution because occured hydrolysis and to avoid the compotition in compound other it the function of KOH for get piperine from concentration extract where in the extract exist another compound when added KOH alkoholis and caused piperine in extract react become salt acid piperate and to eliminated other compound because in the extract still contain purity compound. Purity compound cause piperine compound is alkaloid compound from amida that sustain hydrolisis reaction. So, alkoholis of KOH for isolated piperine in salt is piperate acid. Then solution will stir and filter. Filtering process to filtrate can be seperated from purity. After that, the filtrate divede 3 part, first part 6ml for piperine reaction, 59ml for piperine isolation and another part for degradation. In isolation of piperine, the extract will concentrated until saturated and easy formed crystall. Then, keep it 1 days and not formed crystall. Then crystalitation with ethanol based on the principle of recriztalitation based the different principle in solubilty in the solvent and temperature. In the room temperature, piperine compound in the crystall and polar properties and soluble in etanol as solvent, then piperine in the filtrate will soluble but purity compound will be left in the residue. In this

process, we didnt get crystall it is because when heated apperantice not attend to see the extract and the extract exhaused vaporated. Reaction
O O N O piperine KOH

+ CH 3 CH 2 OH
ethanol

+
NH

O OH

CH 3 CH 2 OK

. Equation reaction:
1.
C 2 H 5 OH

N O

+
O

N H

+
O O

CH 3 CH 2 O

2.
N H O O
+

CH 3 CH 2 O

+
NH C
+

+ CH 3 CH 2 O

3.

NH

+
C
+

+ CH 3 CH 2 O

NH

+
C

O OH

+ CH 3 CH 2 OK

Piperidine 2. Piperine degradation

piperate acid

Degradation is decompotition of compound become a simple compound. In this thing, piperine degradation to get salt of piperate acid. In this degradation process, extract will be reacted with KOH 10% (same with piperine isolation) then concentrated belong 5minutes, produced dark yellow solution. Then, it mixture reflux belong a hour. This reflux to occurred hydrolysis reaction perfectly. In this

process happen is solvent is KOH filled with piperin in round flask, then heat it. Vapor of solvent condentation by condenser become solvent molecule until down to round flask and will solute back piperine that in the flask until surface and all of by continue. Then suspentition by water to dilute and hydrolysis piperine. Then add HCl 10M to neutralization because hydrolysis occur in base condition and must neutralization. Reaction:
O O OC
-

+
O

HOOC

Salt piperate acid

piperate acid

HCl 10M addition to get proton until formed piperate acid occurred hydrolysis reaction. Next, solution keep silent belong one day then filtered and crystallization with ethanol. This process not crystal formed because apperantice mistaken when reflux and added HCl or water.

3. Reaction of piperine a. KMnO4 reaction This experiment we show that piperine easy oxidate because have double bond. This experiment, alkoholis solution add with HCl dilute to decreasse pH because solution in base condition to get netral solution. Then reacted with KMnO4 to oxidate piperine and produce two layers, yellow in uplayer and turbity in the down layer. It is not same with theory must be purple. It is caused while netralization not universal indicator and only litmus paper and dont know the solution netral or not if only used litmus.

Reaction is

b. Benedict reaction At this experiment to show that piperine have aldehide group that is reducing group. Solution that get from isolation added with HCl diluted to decreasse pH because solution in base condition to get netral solution. Solution reacted with benedict reagent produce green brown solution. It is not same with theory must result brick red, green or yellow. It is not same with theory because piperine still contain purity compound and while netralization not universal indicator and only litmus paper and dont know the solution netral or not if only used litmus. Reaction :

c.Molish reaction The purpose of this experiment to identify existence of epoxy group that sign with furfural ring in piperine. Alkoholis solution from extraction added with HCl dilute to decreasse pH because solution in base condition to get netral solution. Then reacted with Molish reaction and formed yellow solution. It is not same with theory because piperine still contain purity compound and while

netralization not universal indicator and only litmus paper and dont know the solution netral or not if only used litmus. Reaction is
O N O O
HCl N

+
H

O H 3C piperanol CH3

+
O epoksi

piperidine hydrochloride

I.Closing 1. Concusion a. The principle of organic compound from chemical nature specially alkaloids extraction alkaloid in acid solution in water and netral or base compound from mixture to separate with solvent. After this solution base so alkaloid set with extraction in solvent. b. Technique isolation in chemical nature is extraction continue, purifying with method crystalization and purifying step with determine melting point. c. Piperine reaction with KMnO4 to show that piperine easy oxidezed. Piperine reaction with benedict to test for carbonyl group in piperine can be reduced by Cu2+. Piperine reaction with molish reagent to find a ring that piperine furfural in epoxy groups. 2.Suggestion a. Apperantice must be attend when vapor the extraction, b. Laboran must prepare our reagent before experiment.

BIBLIOGRAPHY Bruice, P.Y. 2003.Orgnic Chemistry 4th Edition.New York. Longman Hill. Madhavi, B.B. 2009. Extraction, Identification, Formulation and Evaluation of Piperine in Alginate Beads. India. International Journal of Pharmacy and Pharmaceutical Sciences. McMurry, John. 2008. Organic Chemistry 7th edition.USA. Thomson Learning, Inc. McMurry, Susan. 2008. Study Guide and Solutions Manual Fundamental of Organic Chemistry. USA. Thomson Learning, Inc. Tim Dosen. 2012. Penuntun Organik 2. Makassar.Universitas Negeri Makassar. Wood, A.B. 1988. Piperine Determination in Pepper (Pipper ningrum L.) and its oleorensis A reversed phase High-performance Liquid chromatographic Method. England. Flavour and Fragrance Journal.

Answer Question 1.Using potassium hydroxide to remove resin(said to contain chavicine an isomer of piperine. 2. Degradation of piperine
O O OC
-

+
O

HOOC

Salt piperate acid 3. Reaction of Piperine a. Reaction with KMnO4

piperate acid

b. Reaction with benedict

c.Reaction with Molish

O

HCl
N O O

+
H

O H 3C piperanol CH3

+
O epoksi

piperidine hy drochloride