BVB221 Nature’s Pharmacy

Experiment 2: Isolation of Piperine from Black Pepper (Alkaloids)

Introduction

Commercially available black pepper is the dried, full grown but unripe fruit of the perennial
woody vine Piper nigrum. The time of harvest and post-harvest treatment influence peppercorn
colour and flavour (e.g., white pepper is obtained from the dried ripe fruit by removing the outer
portion of the pericarp after soaking in water). Pepper derived from the Piper genus should not
be confused with bell peppers or chilli peppers which are of the Capsicum genus.

Black pepper is known to have a variety of physiological activities:

 Carminative (a remedy for flatulence)
 Diaphoretic (an ingested substance that induces perspiration).
 Diuretic (a chemical that increases the excretion of urine)
 Lipolytic (enhances breakdown of fat)
 Insecticidal

Folk medicine effects of pepper include its use as a carminative, stimulant and tonic, as well as
for treatment of various cancers. In addition to these uses, there are Chinese folklore reports of
white pepper involved in the treatment of malaria, stomach ache, and cholera.

The flavour, aroma, and ‘heat’ of pepper derive from the mixture of volatile oils (mostly mono-
and sesquiterpenes) and alkaloids it contains. These compounds are primarily responsible for the
physiological activity of pepper. The pungency (heat) of pepper is primarily due to the presence
of piperine and its related alkaloids (Figure 1) that are present at between 5-10% w/w (dry
weight).

Alkaloid extraction is usually based on

 Solubility in a particular solvent or solvent combination
 Acid-base extraction: manipulation of alkaloid solubility by adjusting pH

The classical method for isolating piperine from black pepper involves ethanol extraction
followed by overnight precipitation of piperine from 10% alcoholic potassium hydroxide.
Because of the relatively short time available, an alternative method involving extraction into
dichloromethane, precipitation via trituration of the crude alkaloids with diethyl ether, and re-
crystallisation in hot 3:2 acetone:hexane will be used. The present experiment introduces
students to the techniques of refluxingi, triturationii, and suction filtration. The resulting crude
piperine will be purified by recrystallization in the practical in Week 5 and analysed by thin layer
chromatography (TLC).

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Figure 1. Piperine and related alkaloids found in black pepper

Figure 2. Reflux Apparatus (a) disassembled and (b) assembled.

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Procedure (work in pairs)

Part A - Extraction

1. Assemble the reflux apparatus (Figure 2). An assembled reflux apparatus is available for
your inspection.

2. Obtain a sample of ground peppercorns. This will contain 20 g of freshly ground pepper.

3. Add the entire contents of the pepper sample (20 g) to a 250 mL boiling flask.

4. Add 40 mL of dichloromethane (DCM) to the boiling flask. Remember to include a few

boiling chips (anti-bumping granules).

5. Place the boiling flask on the mantle heat and gently raise the support to connect the flask
with the condenser.

Note: Only gentle pressure is required to achieve an adequate seal between the flask and
condenser.

6. Turn the mantle heater on to a LOW setting (2 to 3).

Note: the boiling point (bp) of DCM is 39.6 C. The time required for the solvent reach bp is
relatively short.

7. Open the water chiller valves to commence circulation of water through the condenser.

8. Monitor the flask for indications the solvent is close to boiling. Make fine adjustments to the
heater settings to achieve a gentle boiling/simmering action.

Note: if you need to stop the boiling action quickly, lower the mantle heater away from the flask.

9. Allow the boiling to continue for 20 min. If the condenser is working efficiently, there
should be very little/no smell of DCM.

10. After the required reflux period, turn off the mantle heater and lower it away from the flask.
Allow the reflux apparatus to cool for 5 min. Maintain water circulation through the
condenser while the apparatus is cooling.

Part B – Filtration and concentration

1. Vacuum-filter the pepper/DCM mixture with the aid of a Buchner funnel, filter flask,
rubber adapter and filter paper (Figure 3). While you’re decanting liquid from the boiling
flask into the filter, leave the bulk of the pepper grounds in the flask. This is to allow the
grounds to be “washed” with additional DCM (see next step).

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2. Add 20 mL of DCM to the remaining grounds. Swirl the flask gently, then filter the
contents into the same vessel as the initial filtrate. You will now have approximately 60 mL
of DCM/pepper extract. Remove 2 or 3 drops of crude extract and place it in a labelled
capped vial for analysis by thin-layer chromatography (TLC) in Week 5. Label your
samples as ‘crude’.

3. Transfer the remainder of the extract to a clean 100 mL round-bottomed flask and remove
the DCM using a rotary evaporator. Ask a lecturer/demonstrator/technician to assist you
with using the rotary evaporator. When the extract forms olive-brown viscous oil, sufficient
DCM has been removed to move on to the next step.

Figure 3. Vacuum filtration using a Büchner funnel

Part C - Trituration

1. Cool the extract in an ice bath and add 12 mL of cold ether (diethyl ether). Gently swirl the
solution for 2–3 min. Some piperine precipitate may be visible at this point.

Note: The first ether step assists in the removal of any remaining DCM. That is, ether and DCM
form an azeotropic mixture.

2. Remove ether/DCM using the rotary evaporator.

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3. Cool the extract in an ice bath and add 12 mL of cold ether. Gently swirl the solution. Some
pale yellow piperine crystals should be visible at this point. Allow the flask to cool for an
additional 10 minutes with occasional swirling.

4. Using a clean Büchner funnel, filter flask, and filter paper, collect the straw-yellow crystals
of crude piperine via vacuum-filtration (Figure 3). Wash the crystals twice with 2 mL
aliquots of cold ether. Remove 2 or 3 drops of filtrate and place it in a labelled capped vial
for analysis by TLC in Week 5. Label your sample as ‘filtrate’.

5. Carefully transfer the crude piperine crystals to a labelled sample vial. Label your sample
‘semi-pure’. You will complete the purification of piperine and assess the effectiveness of
the purification protocol in the practical in Week 5.

Questions

 What amino acid pathway provides the substrate(s) for piperine biosynthesis?

 Is piperine a true, pseudo, proto, or false alkaloid?

 What was your % yield of crude piperine (fresh weight basis)?

i
Reflux is a variation on distillation except the condensate is returned to the boiling flask (see Figure 2). The value
of such a process may seem questionable but the technique does offer several advantages;
 Constant temperature: the boiling flask is open to the atmosphere; hence, there is no increase in pressure
within the vessel. The boiling point of the solvent will remain constant.
 Recycling of solvent: Even with long procedures there is no need to add more solvent or be concerned the
boiling flask will run dry.
 Mixing: The constant boiling action continually mixes the sample, enhancing the extraction process
ii
Trituration is a process used to purify crude compounds containing soluble impurities. The desired product is
insoluble in the chosen solvent and the undesired by-products are very soluble or vice versa. The crude material is
washed with the solvent and filtered, leaving the purified product in solid form and impurities in solution