Piperine—The Bioactive Compound of Black

Pepper: From Isolation to Medicinal Formulations

Leila Gorgani, Maedeh Mohammadi, Ghasem D. Najafpour, and Maryam Nikzad

Abstract: Piperine is the major bio-active component of pepper, which imparts pungency and biting taste to it. This
naturally occurring alkaloid has numerous demonstrated health effects and beneficial therapeutic properties; nevertheless,
its biological applications are limited due to its poor solubility in aqueous environments. This emphasizes an implemen-
tation of advanced extraction approaches which could enhance the extraction yield of piperine from pepper and also the
development of new formulations containing piperine to improve its in vivo bioavailability. This paper presents a review on
the therapeutic and medicinal effects of piperine, its isolation from pepper fruit and the development of new formulations
for its medicinal (pharmaceutical) applications. A thorough review on conventional and advanced separation techniques
for the extraction of piperine from pepper is presented and an outline of the most significant conditions to improve the
extraction yield is provided and discussed. Different methods used to measure and quantify the isolated piperine are also
reviewed. An overview of biotechnological advancements for nanoparticle formulations of piperine or its incorporation
in lipid formulations, which could enhance its bioavailability, is also presented.
Keywords: bioavailability, biological activity, extraction, pepper, piperine, nanoparticles

Introduction although some reports pointed to higher piperine content of black

Herbs and spices have a long-standing history of use in culinary
and medicinal preparations. The delightful flavor and health ben-
efits of spices have made them indispensable ingredients in food
processing. Moreover, they have found positions in preparations
of numerous medicines due to their beneficial pharmacological
properties. Among the spices, pepper has occupied a supreme
and unique position due to its characteristic pungency and flavor.
Black pepper, rightly nicknamed the King of Spices, is the most
important and the most extensively consumed spice worldwide.
It is the only spice which is invariably served at dining tables and
is an inevitable ingredient of many prepared foods. Black pepper
has been used for many purposes in the past, continues to be so
currently, and will be expected to remain so in the future.
The value of pepper is owed to its pungency and flavor, which
is attributed to the presence of a naturally occurring alkaloid,
known as piperine, as well as volatile essential oils. The volatile
oils, which constitute about 0.4% to 7% of black pepper (Peter
2006), are responsible for the aroma of pepper, although piperine,
as the major constituent of pepper oleoresins, imparts pungency
to it (Parthasarathy and others 2008). The amount of piperine
varies in plants belonging to the Piperaceae family; it constitutes
2% to 7.4% of both black pepper and white pepper (Piper nigrum
L.; Ravindran 2003; Peter 2006; Parthasarathy and others 2008),
CRF3-2016-1265 Submitted 8/6/2016, Accepted 11/2/2016. Authors are with of pepper was found to be small. The chemical structures of piper-
Biotechnology Research Laboratory, Faculty of Chemical Engineering, Babol Noushir- ine and its analogs are depicted in Figure 2 (Ravindran 2003; Peter
vani Univ. of Technology, 47148, Babol, Iran. Direct inquiries to author Mohammadi
(E-mail: m.mohammadi@nit.ac.ir).
pepper up to 9% (Gaikar and Raman 2002; Agarwal 2010), 4%
of long pepper (Piper longum L.) fruits, and 4.5% of Balinese long
pepper fruits (Piper retrofractum Vahl; Gaikar and Raman 2002).
The piperine content of pepper can be influenced by many envi-
ronmental factors including climate, growing conditions, and its
place of origin (Peter 2006).
Piperine, as the most abundant alkaloid in pepper, was 1st iso-
lated from the extract of pepper by Hans Christian Ørsted in 1819.
It was extracted as a yellow crystalline compound with a melting
point of 128 to 130 °C. The chemical structure of piperine was
later identified as piperoylpiperidine, with the chemical formula of
C17 H19 NO3 , and with the IUPAC name 1-(5-[1,3-benzodioxol-
5-yl]-1-oxo-2,4-pentadienyl) piperidine. Piperine was found to
be a very weak base, which upon acid or alkali hydrolysis decom-
poses to a volatile basic piperine, known as piperidine (C5 H11 N),
and piperic acid (C12 H10 O4 ; Pruthi 1999; Agarwal 2010). 1-
Piperoylpiperidine (piperine) exists as 4 isomeric structures: piper-
ine (trans-trans isomer), isopiperine (cis-trans isomer), chavicine
(cis-cis isomer), and isochavicine (trans-cis isomer), as illustrated in
Figure 1; however, the 3 geometric isomers of piperine have al-
most no pungency (Ravindran 2003). Later investigations have
demonstrated the presence of other alkaloids, including pipera-
nine, piperettine, piperylin A, piperolein B, and pipericine, all
possessing some degree of pungency in the pepper extract. Nev-
ertheless, the overall contribution of these alkaloids to pungency
2006). In spite of controversy over the nature of the compounds
responsible for pungency of pepper, piperine is considered the


C 2016 Institute of Food Technologists®

doi: 10.1111/1541-4337.12246 Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 1
Piperine isolation from pepper . . .
H
H
C O
O N
H H
C C N C
C C C C
H H O H
Piperine
H
O C H
H C O H
C C
H C C
O H C N
Chavicine
Figure 1–Structure of piperine and its isomers.
principal pungent one of pepper as it constitutes around 98% of
the total alkaloids in pepper (Hirasa and Takemasa 1998) and; thus,
the piperine content is taken as a measure of total pungency of
pepper (Parthasarathy and others 2008).
In ancient Chinese and Indian medicine, black pepper was used
as a natural medicinal agent for the treatment and alleviation of
pain, chills, rheumatism, influenza, muscular pains, chills, and
fevers. In tea form, black pepper was also credited for relieving
migraine headaches, strep throat, poor digestion, and even coma
(Parthasarathy and others 2008). It was also used for enhancing
the circulation of blood, increasing the flow of saliva, and stim-
ulating appetite (Pruthi 1993). Recent investigations have shown
that piperine has chemopreventive and antioxidant activities. It
also has immunomodulatory, anticarcinogenic, stimulatory, hep-
atoprotective, antiinflammatory (Darshan and Doreswamy 2004),
antimicrobial (Yang and others 2002), and antiulcer activities (Bai
and Xu 2000). Piperine also has biotransformative effects and can
enhance the bioavailability of different drugs such as rifampicin,
sulfadiazine, tetracyline, and phenytoin by increasing their ab-
sorption, by slowing down the metabolism of the drug, or by a
combination of the 2 (Atal and others 1985; Wu 2007). Piperine
shows a protective effect against radiation and so it can be applied
to cancer patients before radiotherapy (Raman and Gaikar 2002b).
It has also been reported that piperine remarkably increases pan-
creatic lipase activity and stimulates pancreatic amylase, trypsin,
and chymotrypsin (Platel and Srinivasan 2000). Recent evidence
suggests that piperine might play an important role in the reduc-
tion of blood cholesterol, triglycerides, and glucose (Mueller and
Hingst 2013). Some of the traditional uses of pepper and recent
studies on therapeutic effects of pepper/piperine are summarized
in Table 1 (Srinivasan 2007; Meghwal and Goswami 2013).
Despite excellent therapeutic properties of piperine, it is slightly
soluble in water (40 mg/L at 18 °C; Vasavirama and Upen-
der 2014). The low solubility of piperine in water and its poor
2 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
H
H
C O
O
H O C
C
C H
H
Isopiperine
H H
C C N
C C
H O
O Isochavicine
C O
H
H
dissolution is the rate-controlling step in the absorption process
of piperine. The pharmaceutical activities of piperine are limited
due to its low water solubility and because use of it at high con-
centrations can be toxic for the central nervous and reproductive
systems (Veerareddy and others 2004; Pachauri and others 2015).
Attempts have been made to develop new formulations to improve
the bioavailability of piperine. Use of modern nanotechnology for
nanoparticle formulations of piperine and its encapsulation in lipid
matrixes are the most recent advancements to overcome its low
solubility in water.
This paper provides a review on piperine, the bioactive com-
pound of pepper, and its therapeutic properties. The work focuses
on methods used to extract piperine from pepper, with a detailed
review on the most important parameters in the extraction process.
The paper also presents recent approaches for the enhancement of
bioavailability of piperine.
Therapeutic Effects of Piperine
Antioxidant activity
Exposure to radiation and environmental pollutants, tissue in-
jury, infections, and autoimmune processes can lead to the produc-
tion of free radicals (Bagchi and Puri 1998). Free radicals can cause
damage which can be alleviated by increasing the concentration
of antioxidants in tissues. Piperine has antioxidant activity and can
reduce thiobarbituric acid-reactive substances and maintain su-
peroxide dismutase, catalase, glutathione peroxidase, glutathione-
S-transferase, and glutathione levels; it also decreases high-fat-
diet-induced oxidative stress in the cells (Vijayakumar and others
2004). Piperine has liver-protective activity due to its antioxidant
activity. Experiments have shown that piperine reduces both in
vitro and in vivo lipid peroxidation and prevents the decrease of
glutathione (GSH) and total thiols. GSH conjugates xenobiotics
which are eliminated by further glucuronidation (Kaul and Kapil
1993). Piperine has shown a significant hepatoprotective effect on

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Piperine isolation from pepper . . .

O
H H H H H H H
H C
C C N H C C C N
C C C O C C C C
O
H H
C H O H H H O
H
O
Piperanine Piperettine

H
H
C O
O O
H H
H H C H2 H2 H2
H C C C C N
O C C C C C
C C N H2 H2 H2
C C C H O

H H O
Piperolein B
Piperylin A

H2 H2 H2 H2 H2 H2 H
C C C C C C C
H3C C C C C C C
H2 H2 H2 H2 H2 H2 CH
HC CH

C NH
O CH3
Pipericine H2C CH

CH3

Figure 2–Structure of piperine’s analogs.

acetaminophen-induced liver damage in mice due to its radical- Antiinflammatory activity

capturing ability (Sabina and others 2010). Pepper and its main
bioactive compound, piperine, enhance the activity of biotrans-
formation enzymes in the liver in a dose-dependent manner and,
thus, play a chemoprotective role (Singh and Rao 1993). Orally
administered piperine can have a stimulating effect on the digestive
enzymes of pancreas and intestines; and it also increases bile acid
secretion (Ulbricht and others 2008).
Piperine has anticancer and antitumor activity. The antitumor
activity of piperine can be due to its immunomodulatory proper-
ties, including the activation of cellular and humoral immune re-
sponses (Sunila and Kuttan 2004). It has been reported that piper-
ine decreased lung metastasis induced by B16F-10 melanoma cells
by the activation of antioxidative protection enzymes and mod-
ulating lipid peroxidation (Pradeep and Kuttan 2002). The anti-
invasive effects of piperine on fibrosarcoma cells have also been
reported (Hwang and others 2011). Pretreatment with piperine
increased sensitization of HER2− overexpressing breast cancer
cells to paclitaxel-induced growth inhibition and apoptosis (Do
and others 2013). Angiogenesis is a hallmark of tumor progres-
sion and is therefore considered as an important target for cancer
treatment. Piperine inhibits the angiogenic process in vitro and ex
vivo; it also has been shown to exhibit inhibition of breast can-
cer cell-induced angiogenesis in vivo (Doucette and others 2013).
Reports have shown that in a nude mice model xenotransplanted
with prostate cancer cells, piperine treatment remarkably decreased
androgen-dependent and -independent tumor growth (Samykutty
and others 2013).
The pathophysiological response of living tissues to injuries is
referred to as inflammation, which causes local accumulation of
plasmatic fluid and blood cells. It is a defense mechanism that
has evolved in higher organisms to protect them against injuries
and infections; however, the complex events and mediators which
take part in the inflammatory reaction can induce or sustain the
development of many diseases or even aggravate them. For the
therapeutic treatment of inflammation-mediated diseases, the use
of antiinflammatory agents is effective. Antiinflammatory property
refers to the ability of a substance or treatment to reduce inflam-
mation or swelling (Sosa and others 2002). Standard piperine, as
well as hexane and ethanol extracts of piper nigrum L., have revealed
remarkable analgesic and antiinflammatory activity (Tasleem and
others 2014). The antiinflammatory activities of piperine have
been confirmed in many rat models (Mujumdar and others 1990).
Piperine also has shown antirheumatic effects in animal models
and antiinflammatory effects on interleukin 1β (IL1β)-stimulated
fibroblast-like synoviocytes (FLSs; Bang and others 2009). It in-
hibits LPS-induced endotoxin shock by the inhibition of type 1
IFN production, which makes piperine a useful gastrointestinal
antiinflammatory agent (Bae and others 2010). Asthma is an in-
flammatory disease caused by irregular immune responses in the
airway mucosa, with symptoms such as inflammation in the airway,
extreme airway mucus production because of goblet cell hyperpla-
sia, and an increase in the thickness of the airway wall. Piperine has
shown deep inhibitory effects on airway inflammation in a murine
model of asthma due to suppression of Th2 cytokines (IL-4, IL-5,


C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 3
Piperine isolation from pepper . . .
Table 1–Traditional uses of pepper and recent studies on the therapeutic effect of pepper/piperine.
IL-13), immunoglobulin E, eosinophil CCR3 expression, and by
enhanced TGF-b gene expression in the lungs. Therefore, it can
be considered as a possible immunomodulator by downregulating
Th2 cytokines (Kim and Lee 2009).
Bio-enhancing ability
Bio-enhancers are drug facilitators and in combination with
drugs they can enhance the activity of drug molecules through
different routes by increasing the bioavailability of a drug across
the membrane, increasing the effect of drug by conformational
interaction and acting as a drug receptor (Patil and others 2011).
Piperine can enhance the bioavailability of many drugs and can be
applied as a bio-enhancer. The absorption of piperine across the in-
testinal barrier is very fast. Studies have indicated that piperine has
a passive diffusion mechanism, a high apparent permeability coef-
ficient, and short clearance time (Khajuria and others 1998). Be-
cause of the nonpolar nature of piperine, it can regulate the mem-
brane dynamics by interacting with lipids and hydrophobic parts of
4 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
the protein, which modify enzyme conformation due to a decrease
in the property of membrane lipids to act as steric constraints to
enzyme proteins. Piperine can enhance the permeation through
the epithelial barrier because it can induce changes in membrane
dynamics and permeation features, along with induction in the
combination of proteins related to the cytoskeletal function which
increases the absorptive surface of the small intestine (Khajuria and
others 2002). Experiments have shown that piperine improved the
bioavailability of β-lactam antibiotics such as cefotaxime sodium,
amoxicillin (Hiwale and others 2002), and ampicillin and other
types of antibiotics like norfloxacin (Janakiraman and Manavalan
2008). The inhibitory effect of piperine on enzymes which are
responsible for the metabolism of these antibiotics in the liver can
be the reason for their improved bioavailability. Combinations of
the antibiotic rifampicin with piperine considerably enhanced the
inhibitory effect on Mycobacterium smegmatis as compared to ri-
fampicin alone (Balakrishnan and others 2001). It has also been
shown that piperine can enhance the pharmacokinetic effects of

C 2016 Institute of Food Technologists®
Piperine isolation from pepper . . .

the gatifloxacin, an antibacterial agent, in laying hens by inhibitingfinal product cost. Thus, selection of a suitable extraction method
the enzymes responsible for the metabolism of gatifloxacin in the and extraction solvent profoundly affects the economy of the pro-
liver (Patel and others 2011). Docetaxel is a cytotoxic chemother- cess. Solvent extraction by aliphatic and chlorinated hydrocarbons
apeutic agent and recently has been used as the most impor- is among the conventional methods used for piperine extraction
tant treatment for metastatic castration-resistant prostate cancer (Raman and Gaikar 2002b). Nevertheless, these solvents are not
(CRPC). The antitumor effect of docetaxel in a xenograft model selective toward piperine and, thus, the extract obtained by this
of human CRPC was improved by co-administration of piper- method always contains some major components such as resins
ine with docetaxel (Makhov and others 2012). Piperine was also and gums. For pharmaceutical applications, the purity of piperine
applied for enhancing the bioavailability of acyclovir. The emulsi- should be 95% to 98%. Therefore, the oleoresin extract with 40%
fication solvent evaporation method was used to prepare acyclovir- to 50% purity requires further purifications. The most common
loaded floating microspheres and the effects of adding piperine on method for purification of piperine are crystallization from aque-
the bioavailability of acyclovir was investigated. Results showed ous alcoholic solutions and treatment with aqueous alkali solutions
that using microspheres containing piperine increased the rela- which inevitably reduce the piperine yield (Leonard and others
tive bioavailability of acyclovir compared to the drug solution or 1960). There are many types of solvents used for piperine extrac-
piperine-free microspheres (Khatri and Awasthi 2016). tion including dichloromethane, petroleum ether, diethyl ether
Piperine also increases the bioavailability of herbal and con- (Epstein and others 1993; Kanaki and others 2008), alcoholic sol-
ventional drugs such as resveratrol and curcumin (Mueller and vents like ethanol, hydrotrope solutions, and ionic-based solutions
Hingst 2013). Studies have shown that piperine remarkably in- (Meghwal and Goswami 2013).
creased the in vivo bioavailability of resveratrol by inhibiting its Traditional solvent extraction methods include soaking, macer-
metabolism and decreasing the required dose of resveratrol in a ation, and Soxhlet extraction. These methods usually require long
clinical setting (Johnson and others 2011). Piperine also increased extraction time and/or high temperature which impose the risk
the bioavailability of curcumin and enhanced its protective ef- of thermal degradation of thermo-sensible bio-active compounds
fects against chronic unpredictable stress (CUS)-induced cognitive (De Castro and Garcıa-Ayuso 1998). Moreover, use of a large
disorder and associated oxidative damage in mice. Bioavailability- amount of solvent and poor extraction selectivity can add to the
enhancing of curcumin could be due to preventive effect of piper- disadvantages of conventional extraction techniques. Such draw-
ine on the intestinal and hepatic metabolism of curcumin (Rinwa backs have motivated researchers to look for enhanced extraction
and Kumar 2012). It was reported that curcumin-piperine (Cu-Pi) methods to minimize product loss and produce bio-active com-
dual-drug-loaded nanoparticles were able to overcome low oral pounds with specific quality characteristics. The modern tech-
bioavailability of curcumin and cancer cell targeting limitation niques for extraction of piperine are supercritical carbon diox-
in cancer treatment (Moorthi and others 2012). Curcumin with ide (CO2 ) extraction, ultrasound-assisted extraction (UAE), and
piperine-loaded cubosome nanoparticles also improved the oral microwave-assisted extraction (MAE), which are extensively dis-
bioavailability and tissue distribution of curcumin (Tu and others cussed in the following sections. An overview of the conventional
2014). and modern extraction methods is provided here; although some
of these techniques are quite potential to extract piperine with
Isolation of Piperine from Pepper high purity and enhanced yield, they are still underutilized and
Black and white peppers are composed of 2 main components; few works have been published in their regards. A scheme of iso-
namely, volatile (essential) oil and pungent compounds, which are lation of piperine from pepper using different extraction methods
responsible for their aroma and pungency, respectively. Pepper oil, is presented in Figure 3.
which is normally extracted by steam distillation of dried pepper
corns, does not contain pungent compounds and only represents Soxhlet extraction
aromatic and odorous constituents. Because of its aroma, pep- Use of the Soxhlet method for extracting valuable bio-active
per oil is highly valued in the fragrance industry and is used in compounds has a long history. Although outdated, it is still used
high-grade toiletry products and the perfume industry as well as as the reference leaching technique to evaluate the efficiency of
the flavor industries (Nair 2011). However, pepper is highly es- newly developed extraction methods. For the Soxhlet extraction,
teemed as a condiment, around the world, for its pungent and a defined amount of dry sample is placed in a thimble made of
nonvolatile compounds which are found in the oleoresin of pep- filter paper which is then placed in a designated distillation system
per. Oleoresin is the solvent-extractable portion of pepper which containing the desired extraction solvent. The solvent is heated
constitutes around 6% to 13% of black pepper (Ravindran 2003) and the generated vapor is condensed which then drips into the
and possesses odor, flavor, and pungency. It is obtained by re- thimble until the solvent reaches to the overflow level. At this
peated extraction of ground pepper by volatile organic solvents, time, the solution of the thimble-holder which carries extracted
such as ethanol, acetone, ether, dichloroethane, or ethyl acetate, solutes flows back into the distillation flask. Although the solute
and subsequent removal of solvent under reduced pressure to trace remains in the distillation chamber, solvent passes back into the
levels. The organoleptic properties of the so-called oleoresin are solid bed. This process continues until the bio-active compound
determined by its volatile oils and piperine contents whose abun- is fully extracted (Huie 2002; Azmir and others 2013).
dance depends on the variety of pepper and its maturity stage, In Soxhlet extraction, the sample is frequently in contact with
the used extraction solvent, and the extraction condition (Pruthi fresh solvent which accelerates mass transfer and extraction of the
1999). Typically, the oleoresin offered by major producers for sale solute. As another advantage, use of filtration after the leaching step
is reported to contain 15% to 20% volatile oil and 35% to 55% is eliminated in Soxhlet extraction. Although the basic equipment
piperine (Nair 2011). of Soxhlet extraction is simple, it requires a considerable amount of
Generally, an ideal extraction method should be comprehensive, solvent. The most important disadvantages associated with Soxhlet
rapid, simple, and cheap (Benthin and others 1999). In some cases, extraction are lengthy extraction time, solvent loss, and damage to
the cost of extraction and purification steps is almost 50% to 90% of the environment (De Castro and Garcıa-Ayuso 1998).


C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 5
Piperine isolation from pepper . . .

Figure 3–The scheme of piperine extraction from pepper using different methods.

Subramanian and others (2011) used a double-bypass Soxhlet or water-insoluble organic materials in aqueous solutions (Raman
apparatus (DBSA) for the extraction of piperine from Piper nigrum and Gaikar 2002a). It is a result of the tendency of amphiphilic
and compared its performance to that of conventional Soxhlet. hydrotrope molecules for self-aggregation or possible accumula-
The implemented DBSA was a modified Soxhlet apparatus which tion around other hydrophobic molecules (Bhat and Gaikar 1999).
had a double bypass side-arm instead of one bypass side-arm avail- These accumulations are likely very smaller than micelles of surfac-
able in the conventional Soxhlet extractor. It was hypothesized thattants and much less cooperative. In aqueous solutions, hydrotropes
the developed DBSA could decrease the extraction time and en- show a considerable feature of destroying the lamellar crystalline
hance the extraction cycle. The achieved results approved such structure of surfactants, producing a continuous isotropic liquid
premise when a piperine extraction yield of 3.9% was attained solubility zone (Meireles 2008). Another differentiating character
in 12 h with a cycle time of 8 min using DBSA, although the of hydrotropes, compared to surfactants, is their ability to distin-
extraction yield of 3.8% was obtained within 22 h and cycle time guish between different organic compounds of a mixture, even
of 16 min using the conventional Soxhlet apparatus. Rajopadhye nearly related substances (Raman and Gaikar 2002a). It is this ca-
and others (2012) extracted piperine from the root of Piper ni- pability of molecular diagnostics that is useful for the enhanced
grum by Soxhlet extraction with methanol in 8 h and reached a extraction of a substance from naturally occurring crude materi-
piperine content of 9.56 ± 0.83 mg/g of root. Yamaguchi and als. The high solubilization power of a hydrotrope should promise
others (2011) extracted piperine from black pepper corns by 3 great extraction capacities for insoluble organic active compounds.
methods, namely, solvent extraction, Soxhlet extraction, and su- A good hydrotrope is one which has high water solubility and, at
percritical carbon dioxide extraction. In solvent extraction, pepperthe same time, maintains its hydrophobicity. In fact, it is the balance
was mixed with methanol, sonicated for 10 min and then filtered. between these 2 counteracting characteristic which determines the
The time of Soxhlet extraction was 20 h and methylene chlo- hydrotropic solubilization (Lee and others 2003).
ride was used as the extraction solvent. The total run time for Hydrotropic solutions can extract hydrophobic components
supercritical CO2 extraction was 0.25 h. The amount of extracted from a complex biomatrix by disrupting plant cell structures. Hy-
piperine was 54.9, 52.7, and 56.6 mg/g for solvent extraction, drotrope solutions make the pericarp of Piper nigrum fruits per-
Soxhlet, and supercritical CO2 extraction, respectively. meable thus simplify the selective extraction of piperine. This
extraction process can be explained by possible adsorption of hy-
Hydrotropic extraction of piperine drotrope molecules onto the cellulosic cell wall, destroying its
Hydrotropy refers to the potential of highly water-soluble but structure, and then penetrating into the cell membrane, helping
mild surface-active amphiphilic organic salts named hydrotropes. in disorganizing the amphiphilic lipid bilayer and enabling easy
Hydrotrope additives can enhance the solubility of slightly soluble release of piperine. The extraction agent requires to prevail several

6 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016 
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Piperine isolation from pepper . . .
resistances in the process. Before hydrotrope molecules can access
the dispersed piperine in the cell, they face some resistances in-
cluding penetration into the cellulosic layer followed by diffusion
into the phospholipid bilayer. At enhanced concentrations of the
hydrotrope, a greater osmotic pressure also expands through the
cell wall; therefore, at higher concentrations, a hydrotrope solu-
tion penetrates into the cell matrix gradually. There is an initial
lag time after which hydrotrope monomers diffuse into the cell
wall and undermine the liquid crystalline nature of the bilayer; at
this time the swelling of the cell wall is lost. The aqueous solution
subsequently penetrates into the cellular matrix and piperine is
then transported into the external hydrotrope medium; these are
considered the fast stages (Raman and Gaikar 2002a).
Generally, the extraction rate of piperine depends on the con-
centration of hydrotrope, temperature, and particle size of the
pepper matrix. There is also a distinct relationship between
the length of the hydrotropic chain and extraction efficiency.
Raman and Gaikar (2002a) used sodium n-butyl benzene sul-
fonate (NaNBBS), sodium cumene sulfonate (NaCS), sodium xy-
lene sulfonate (NaXS), and sodium p-toluene sulfonate (NaPTS)
for piperine extraction from black pepper. They observed that
NaNBBS was the most efficient hydrotrope in dissolving piperine;
this ability was attributed to its longer chemical chain. Solubiliza-
tion also enhances with an increase in hydrophobic volume. This
demonstrates that hydrophobic areas existing in the hydrotrope
aggregates provide a microenvironment which is compatible with
the hydrophobic nature of piperine. It is also conceivable that the
presence of a highly hydrophobic solute results in the promotion
of aggregation behavior of the hydrotrope (Balasubramanian and
others 1989).
Increasing the extraction temperature causes more lysis of the
cell and, as a result, the cell wall becomes more permeable to the
hydrotrope solution (Subbarao and others 2012). Disruption and
solubilization of the cellulose polymer inside the cell wall reduces
the contribution of this polymer to the firmness of the cell wall.
Nevertheless, the enhanced cell rupture at high temperatures de-
creases selectivity toward desired bio-active compounds, as extrac-
tion of unfavorable oleoresins is also facilitated at this condition;
this eventually decreases the purity of extract (Mishra and Gaikar
2009). The existence of phosphorus-reducing sugars and amino
acids in the extract phase confirms the premise of breakdown of
cellulose as well as partial degradation of membrane proteins. It was
reported by Raman and Gaikar (2002a) that an extraction temper-
ature of 30 °C is suitable for hydrotropic extraction of piperine;
this temperature was found to be safe enough to only destabilize
the liquid lamellar structure of cell membranes, without disrupt-
ing the cellulose polymer, and to enhance the selective transport
of piperine into the hydrotrope medium. Purity of the extracted
piperine is also found to decrease significantly when the parti-
cle size is reduced. With a decrease of particle size, cell breakage
increases and the hydrotrope solution penetrates more effectively
into the cellular matrix which leads to solubilization of undesirable
solutes along with piperine (Raman and Gaikar 2002a). Raman
and Gaikar (2002a) observed that the purity of extracted piperine
decreases from 98% to 89% with a decrease of pepper particle size
from 710 to 50 μm.
Supercritical fluid extraction of piperine
Supercritical fluids are widely used for extracting compounds
from natural materials. Supercritical fluid extraction (SFE), which
has recently become very popular in the spice and aromatic crops
industries, is a rapid, selective, efficient, and clean method for the

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 7
extraction of natural products. SFE uses solvents above their crit-
ical temperatures and pressures with liquid-like densities which
cause great loading of solutes. This, combined with the pressure-
dependent solvating potential of supercritical fluids, makes them
perfect solvents for separations and reactions (Lang and Wai 2001;
Reverchon and De Marco 2006; Pourmortazavi and Hajimir-
sadeghi 2007). Kumoro and others (2009) showed that solubility
of piperine in carbon dioxide increased at supercritical and near-
critical conditions, and it was deduced that the enhancement in
piperine solubility was mainly attributed to the increase in CO2
density along with an increase of pressure.
Supercritical fluids are very capable to conduct an effective mass
transfer, permitting better and quicker penetration into sample
matrixes and selective extraction of favorable compounds due to
their small viscosities and great molecular diffusivities the same
as gases as well as their small surface tension. The most serious
drawback of SFE, compared to traditional methods, is its higher
investment costs (Lang and Wai 2001; Herrero and others 2006,
2010). However, the overall process (extraction plus separation) is
almost cost-effective and can be simply scaled up to industrial scale.
Liquid carbon dioxide (Tc = 31.1 °C and Pc = 73.8 bar) is the most
common fluid used for SFE because it is clean, noncombustible,
nontoxic, easily available, and of low cost (Mukhopadhyay 2000).
The only disadvantage regarding the use of CO2 as supercritical
fluid is that CO2 has low polarity and might be more suitable for
the extraction of nonpolar compounds. However, this limitation
can be overcome by the addition of chemical modifiers such as
alcohol (1% to 10% of supercritical fluid) to increase the polarity
of the solution (Singh 2014). Supercritical CO2 has been used
as an ideal solvent for pepper extraction wherein around 98% of
piperine and 81% of essential oil could be extracted (Peter 2006).
For successful extraction not only the affinity of the desired and
undesired compounds toward extraction should be considered, but
also mass transfer resistances along the path to the specific location
of the desired compound, which depend on the structure of the
raw material and can play an important role (Reverchon and De
Marco 2006). Temperature is another important parameter which
affects the SFE yield. When temperature increases, the solvent
power decreases due to reduction in the density of supercritical
fluid (Rostagno and Prado 2013). The most important parameter
in SFE is pressure. Increasing the extraction pressure enhances
the solvent power, but it reduces the extraction selectivity. Some
other effective parameters in SFE are the flow rate of fluid, particle
size of the sample subjected to extraction, and extraction duration
(Reverchon and De Marco 2006).
Vitzthum and Hubert (1978) extracted piperine from pepper at
350 atm and 60 °C within 3 h using dry CO2 followed by 2 h
extraction using wet CO2 at the same condition. They obtained
a yellowish semisolid mass with a crystalline constituent. The ex-
traction yield of piperine was 7 wt% and 98% of the piperine
transferred to the extract. In another investigation carried out by
Kurzhals and Hubert (1980), piperine was extracted from pep-
per using a mixture of CO2 and propane with a molar ratio of
58.8:41.2 at 52 °C and 78 bar. After 2 h, 98% of piperine trans-
ferred to the extract. Sovová and others (1995) extracted piperine
from pepper using CO2 at 28 MPa and 40 °C and obtained ex-
traction yields of 6.7 to 7.6 wt%. They also found that the rate of
extraction increased with an increase in temperature because of the
enhancement of piperine solubility in carbon dioxide. An enzyme-
assisted supercritical CO2 extraction was examined by Dutta and
Bhattacharjee (2015) for extracting piperine from black pepper
oleoresin under batch and continuous operations. It was reported
Piperine isolation from pepper . . .
that in the continuous mode, the yield gained by nonenzymatic
supercritical CO2 extraction at 60 °C and 300 bar within 2.25 h
(as optimized condition) was 4.6 ± 0.4 g extract/100 g dry black
pepper (1.36 ± 0.02 mg piperine/g pepper), whereas the corre-
sponding yield with the batch mode under the same condition was
2.1 ± 0.3 g extract/100 g dry black pepper (1.23 ± 0.05 mg piper-
ine/g pepper). In comparison, with the continuous mode of en-
zyme (α-amylase)-assisted supercritical CO2 extraction, the yield
of extraction increased by 15% and reached 5.3 ± 0.4 g extract/
100 g dry black pepper (1.45 ± 0.04 mg piperine/g pepper); this
enhancement was considerably lower than the improvement (53%)
observed in the extraction yield (3.8 ± 0.2 g extract/100 g dry
black pepper and 1.36 ± 0.04 mg piperine/g pepper) in the batch
mode under the same operating condition. It was inferred that the
enzyme needed an incubation time to hydrolyze the starch present
in the black pepper matrix. This incubation time was provided for
the enzyme in batch operations, which resulted in the increase in
the extraction yield of oleoresin from the pepper matrix.
UAE of piperine
Soxhlet extraction and batch solvent extraction are traditional
extraction techniques with disadvantages such as lengthy extrac-
tion time, solvent wasting, and labor intensity. Recently, to over-
come the disadvantages associated with conventional methods,
new extraction techniques have been developed. UAE is one of
these techniques. The mechanism of UAE is based on the cavi-
tation phenomenon and thermal effects, which enhance the mass
transfer through the cell wall. Moreover, the collapse of cavitation
bubbles causes better cell disruption by formation of microjets due
to asymmetrical bubble collapse close to the solid surface (Mason
and others 2011). The frequency, acoustic power, gas atmosphere,
hydrostatic pressure, nature, and temperature of the solvent, and
geometry of the reactor are among the factors which can af-
fect the cavitation (Henglein and Gutierrez 1993). High pressures
(up to 1000 bar) and temperatures (up to 5000 K) are created
due to imploding of these bubbles that can generate high en-
ergy and also some interesting physical effects (Cintas and Luche
1999).These physical and thermal effects cause liquid circulation,
cell wall breakage, decline in particle size, and increased mass
transfer through cell membranes (Yang and Zhang 2008). Con-
sequently, solvent penetration into the plant body improves and
cell breakage occurs (Toma and others 2001) and, as a result, the
extraction yield in UAE will be increased. Thus, UAE has advan-
tages such as better solvent penetration, lower dependence on the
applied solvent, extraction at lower temperatures, lower extraction
time, and higher product yields (Mason 1999; Vinatoru 2001).
These features make the UAE attractive as an enhanced extraction
method and also for scaling-up. It could be applied in an extraction
unit where the plant material directly contacts the solvent.
Type of solvent, extraction time, solid-to-solvent ratio, duty cy-
cle, ultrasound power, and temperature are the factors that affect
the extraction efficiency (Mason and others 2011). Selection of the
most appropriate solvent for extraction of the target compounds
from the sample is a crucial step in any extraction technique, es-
pecially UAE. Polarity index, viscosity, surface tension, and vapor
pressure of solvent are the factors that must be considered in the
selection of a suitable solvent (Sun and others 2011). Low vapor
pressure of solvent causes less bubble formation, but these bubbles
implode with higher force; thus, more of the target compound is
extracted. Since continuous exposure to the ultrasound may lead
to the degradation of the material, it is always recommended not
to use the ultrasound in a continuous mode; therefore a duty cycle
8 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
is determined for the system, based on which the ultrasound appa-
ratus is turned on and off. Large amplitude waves move across the
liquid medium and the bubbles collapse more explosively when
the ultrasound power is enhanced (Yang and others 2013). Tem-
perature affects the solubility, rate of transfer of target compound
to the solvent, and also cavitation phenomenon; both the cavita-
tion and thermal effects display significant influences on extraction
yield. Cavitation is responsible for the implosion of cavitation bub-
bles, which causes great agitation. The thermal effect is responsible
for the greater solubility of solute and improvement in mass trans-
fer due to a decrease of solvent viscosity (Entezari and Kruus
1996; Raso and others 1999). In an attempt to use UAE, Rathod
and Rathod (2014) used 3 different solvents (acetone, ethanol,
and hexane) for extracting piperine from Piper longum, and they
observed that acetone gave the maximum yield (4.53 mg/g), fol-
lowed by ethanol (4.32 mg/g), and hexane (4.08 mg/g). Although
acetone and ethanol approximately have the same polarity index,
higher yield was obtained using acetone due to greater diffusion
into the solid matrix owing to the lower viscosity of acetone.
Since the distinction in extraction yield obtained by acetone and
ethanol was not remarkable in their experiment, and considering
handling difficulty and the price of acetone, ethanol was selected
as the preferred solvent. The authors experimented with 1:2.5
to 1:40 solid-to-solvent ratios and observed that the extraction
yield enhanced with a decrease in solid-to-solvent ratio towards
1:10. They also found out that the piperine extraction yield grew
logarithmically up to 18 min, and afterwards a slow increase was
observed with extended extraction time. An enhancement in ex-
traction yield was observed when the duty cycle was increased
until the equilibrium was attained at a duty cycle of 80% (48 s
on, 12 s off). The ultrasound power of 125 W and temperature
of 50 °C were also reported as suitable operation condition. At
the optimized condition, maximum extraction yield of 5.8 mg
piperine/g black pepper was obtained.
Ionic liquid (IL)-based extraction of piperine
Use of ILs as reaction media has increased during the last 2
decades (Jessop 2011; Angell and others 2012). ILs are molten
salts, composed of anions and cations with low melting points
of typically less than 100 °C (Wasserscheid and Welton 2008).
ILs have some unique properties over traditional solvents: low
vapor pressure, high chemical and electrochemical stability, and
high polarity (Wasserscheid and Welton 2008; Aparicio and oth-
ers 2010). The physicochemical properties of ILs, which depend
on their structures, have a great effect on the extraction efficiency
of the target analyte (Cao and others 2009). The nature of the
ions and their interactions directly affects the physicochemical,
thermal, and solvent properties (Huddleston and others 2001). ILs
have been widely used for extracting bio-active compounds from
natural materials, and it is believed that the hydrophobic interac-
tion between aqueous ILs and the bio-active compounds such as
piperine (Cao and others 2009), curcumin (Xu and others 2015),
tannin (Chowdhury and others 2010), rutin, and quercetine (Wu
and others 2012) is the main driving force for effective extraction.
Recently, the extraction method has been combined with
UAE (ILUAE) to offer an enhancement with several advan-
tages over conventional extraction (Chatel and MacFarlane 2014).
The ILUAE technique provides greater extraction, efficiency, and
clearly reduces extraction time compared to common methods.
It is also an energy-efficient technique and has high potential in
“green” chemistry (Chatel and MacFarlane 2014). In this regard,
Cao and others (2009) worked on ILUAE extraction of piperine

C 2016 Institute of Food Technologists®
Piperine isolation from pepper . . .
from white pepper. They applied 1-butyl-3-methylimidazolium
(C4 MIM) ILs with 4 different anions (BF− − −
4 ,BF ,H2 PO4 , and
−
PF6 ) and realized that the extraction efficiency for them was in the
order of BF− − − −
4 > Br >H2 PO4 > PF6 because of decreasing hy-
drophilicity of these 4 anions. As alkyl chain length increased from
propyl to butyl, the extraction efficiency significantly decreased.
Moreover, it was found that the groups which substituted on the
alkyl chain had a remarkable influence on the extraction. Because
of changes in the water miscibility and strength of ILs’ hydrogen
bonds, the extraction efficiency of the IL without sulfonic group
on the 1-alkyl chain was nearly 30% more than that with sulfonic
group. Considering these results (C4 MIM; BF4 ) was selected as
the preferred IL. In their experiment, extraction time, concentra-
tion of the (C4 MIM; BF4 ), ultrasonic power, and solid-to-liquid
ratio were considered as the parameters, which could affect the
extraction steps and extraction efficiency. They reported that the
maximum extraction yield was 3.577% which was attained using
(C4 MIM; BF4 ) at a concentration of 2 M and ultrasound power
of 500 W, with a solid-to-liquid ratio of 1:15 after 30 min.
Microwave-assisted extraction of piperine
In the electromagnetic spectrum, microwave radiation lies be-
tween infrared and radio frequencies. Microwaves have electric and
magnetic fields, which swing perpendicularly to one another in
frequencies in the range of 0.3 to 300 GHz. Over the last decades,
microwave technology has witnessed extensive growth in several
areas. Recently, microwave-assisted extraction (MAE) has been ap-
plied for the extraction of volatile organic compounds from many
types of natural materials. In this method, volatile constituents
absorb microwave energy and evaporate from the solid matrixes
and then are recovered by condensation (Chan and others 2011).
MAE is considered a relatively selective extraction technique be-
cause the generated microwave heat acts directly on the molecules
and the targeted material can be selectively heated based on its di-
electric constant. The mechanism of heating by microwaves is ei-
ther dipole rotation or ionic conduction (Eskilsson and Björklund
2000), where the interaction between microwave radiation and
dielectric field of polar molecules or ions generates heat. Any in-
crease in temperature and pressure accelerates MAE because the
extraction solvent can absorb higher amounts of microwave en-
ergy (Wang and others 2008). In conventional solvent extraction,
mass transfer and heat transfer are in different directions, where
mass transfer occurs from the inside to the outside, whereas heat
transfer takes place from the outside to the inside. In contrast, in
microwave solvent extraction, both transport phenomena occur
at the same time from the inside of the material subjected to the
extraction into the bulk of the solvent. Synergistic combination
effects of these 2 transfer phenomena could be one of the reasons
for the enhanced extraction rate observed in MAE (Mason and
others 2011). Another feature of microwave heating is its volu-
metric heating nature wherein heat can be generated throughout
the volume of the material, because microwaves can penetrate the
materials and deposit energy within them. This is unlike the con-
ventional heating in which, heat is transferred from the surface
towards the center of the material. Thus, MAE can be performed
much more rapidly and with higher efficiency as compared to
conventional extraction methods.
When a raw material is subjected to microwave irradiation, the
volatile compounds 1st evaporate within the cell. The accumu-
lation of water and oil droplets and eventually their coalescence
creates a large pressure gradient across the cell membrane. The
aggregation of coalesced bubbles causes swelling and deformation

C 2016 Institute of Food Technologists® Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 9
of cell structure from inside. With progression of this phenomenon
and growth of pressure inside the cell, the internal pressure even-
tually dominants the mechanical resistance of the cell and brings
about cell disruption. At this time, the organic solvent can easily
penetrate into the cell structure and dissolve the targeted com-
pound and then diffuse back into the bulk of the solvent. In fact,
the rupture of the cell wall eliminates the main mass transfer re-
sistance to and from the cell and enhances the rate of transfer of
the desired compounds into the extraction medium. It has been
reported that exposure of dry pepper to microwave irradiation
can increase the temperature to around 300 °C as a result of di-
electric heating of polar constituents inside the cell (Raman and
Gaikar 2002b). This high temperature accelerates the hydrolysis of
cellulose-solvent linkages that can weaken the cell structure and
lead to easier collapse of the cell wall.
Soaking of materials to be extracted in water, or hydration
in the other words, leads to an increase in extraction rate. This
is because of the enhancement of the dielectric permittivity of
biopolymers, along with hydration, which brings about the fast
dielectric heating (Bayley 1951). The dielectric heating of water
inside the pepper cell creates a primary monolayer of water on
protein and lipid molecules which leads to a greater dielectric loss,
which is a measure of the ability of the material to dissipate the
microwave energy as heat, and it improves the heating rate. This
is followed by distribution of the generated heat throughout the
material, through protons and other ionic materials that are ca-
pable of travel around the hydrated molecules (Pethig 1985). In
the case of natural materials, including cellulosic fiber, although
the strength of the cell wall is very huge (82.7 MPa) and cellu-
lose with significant numbers of hydrogen bonds provides great
strength, nevertheless, at temperatures beyond 250 °C some hy-
drogen bonds become unstable and hydrolyze quickly (Grant and
Halstead 1998). Cellulose can then rapidly conduct the generated
dielectric heat within itself or the cell due to its ionic conductivity.
Considering the above discussion, it can be inferred that soaking
of materials in water can be an effective method to enhance the
extraction rate, as the temperature of hydrated molecules within
the cells reaches to levels which are high enough to accelerate the
disruption of the cellulosic cell wall. Raman and Gaikar (2002b)
showed that the extraction rate of piperine was 6-fold higher when
pepper was soaked in water for 4 h prior to MAE. Among the
samples, those which were soaked for a longer time, and which
were more hydrated, displayed much greater rates of extraction in
comparison to those which had a short soaking. The efficiency
of the MAE process depends on the operating conditions and the
parameters which affect the extraction mechanisms and yield. The
nature of extraction solvent is one of the most important factors.
Solubility of a target compound in the solvent, solvent penetration
rate, and its dielectric properties are factors that should be consid-
ered when selecting a suitable solvent for MAE. Solvent toxicity
should also be considered. Polar and polarizable solvents can well
absorb microwave radiation, whereas nonpolar and weak polariz-
able solvents are transparent to microwave radiation and do not
absorb it (Kappe and others 2012). Besides the dielectric heating
occurring in the cell, the heating effect of polar solvents results
in greater destruction to the cell wall and a more enhanced cell
disruption rate. In this case, however, the potential of the hot polar
solvent to diffuse within the cell and dissolve the oleoresins leads
to lower selectivity (Chan and others 2011). Raman and Gaikar
(2002b) worked on piperine extraction from Piper nigrum by MAE.
They examined several extraction solvents ranging from polar to
nonpolar, such as ethanol, dichloromethane, toluene, heptane, and
Piperine isolation from pepper . . .
petroleum ether. Their results showed that nonpolar solvents, such
as petroleum ether, gave the highest extraction efficiency, defined
on the basis of actual piperine content of raw material, of 94% with
a purity of 85%. Although using polar solvents such as ethanol and
moderately polar solvents like dichloromethane, the piperine ex-
traction efficiency was merely 75% to 80%, with a lower piperine
purity of 60% and 72%, respectively. They declared that the higher
extraction yield achieved by nonpolar solvents could be attributed
to their larger penetration depth of microwave radiation, defined
as the depth at which microwave power falls to about half of its
original value at the material surface. The penetration depth of po-
lar solvents is smaller than that of nonpolar solvents, as a significant
amount of microwave irradiation is absorbed by them.
Microwave power and extraction temperature are 2 other im-
portant factors in MAE. During microwave heating, membranes
of oil globules rupture and the oil aggregates very close to the
cell wall. The protein structures also agglomerate into larger par-
ticles. At low microwave powers, the coalescence of fat globules
occurs within a longer period of time, although at higher powers,
the confluence of smaller lipid bodies into bigger agglomerates
takes place at the primary stage of heating, causing quick cell
rupture and an enhanced extraction rate. However, it is worth to
note that high microwave powers cause degradation of thermally
sensitive compounds and, as a result, poor extraction yields are
achieved. Generally, extraction yield increases proportionally to
the power increase, until the increase becomes insignificant or the
yield declines (Xiao and others 2008). As reported by Raman and
Gaikar (2002b), use of high microwave powers of 300 and 450 W
increased solvent loss by 16% to 20%; whereas, at a power level
of 150 W the solvent loss decreased to 8%. Temperature and mi-
crowave power are 2 dependent parameters where an increase
of microwave power increases the extraction temperature. At high
extraction temperatures, the solvent power enhances due to the de-
creases in viscosity and surface tension (Mandal and others 2007).
Solid loading in MAE is also among the parameters which affect
the extraction yield. Piperine extraction decreases with an increase
in solid loading of the suspension. With an increase of solid load-
ing, and thus solid particle concentration, stirring of the solvent
becomes inadequate (Wang and Weller 2006); thus, those particles
which are located close to the container surface and face a greater
extent of radiation will absorb considerably more microwave en-
ergy. In contrast, solid particles which are located in the depth of
the irradiated sample may not absorb the same irradiation dose as
those located on the surface and thus would be affected to a lower
extent by the incident microwave energy. It has been reported that
with increases in solid loading and sample thickness, the effective
extraction of piperine decreases due to the lower effectiveness of
microwave radiation on particles located in the center of the con-
tainer (Raman and Gaikar 2002b). This implies that when solid
loading increases, a higher period of exposure is required to obtain
the similar extent of extraction.
A summary of the implemented extraction methodologies for
extraction of piperine as the bio-active compound of pepper, the
achieved extraction yields as well as benefits and drawbacks of each
method are summarized in Table 2.
Detection of Piperine
There are different quantitative methods for the detection
of piperine: the Kjeldahl method (Winton and Winton 1945),
some colorimetric methods (Graham 1965a, c), gas chromatogra-
phy (GC; Committee 1984), ultraviolet (UV) spectrophotometry
(Singh and others 2011; Vishvnath and Jain 2011), and high-
10 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
performance liquid chromatography (HPLC; Raman and Gaikar
2002a; Rathod and Rathod 2014). In the Kjeldahl method with
which total nitrogen is measured, the estimated amount of piper-
ine would be higher than the real amount as it measures all of
the nitrogenous compounds including piperittine, cavicine, and
some of the free amino acids present in pepper (Ravindran 2003).
In colorimetric methods, reagents which can directly react with
the piperine molecule are used. The 1st method developed relied
on hydrolyzing the methylenedioxy group of piperine, by chro-
motropic acid in the presence of concentrated sulfuric acid, to
formaldehyde upon which a definite color was developed (Graham
1965a). In this method, the test results tend to be high too, as other
methylenedioxy group-containing compounds such as piperittine
will react with the reagent and give a higher false reading (Tainter
and Grenis 2001). The UV spectrophotometric method is based
on piperine absorption in the UV region at 343 nm. This method
is simple and fast and has high specificity for piperine; it does
not tend to capture other isomers of piperine (Singh and others
2011). The GC method separates the alkaloids available in the
oleoresin and also gives some information about the degradation
state of piperine (Noyer and others 1999). Recently, HPLC has
become a more common method for the detection of piperine,
and its accuracy is better than the UV spectrophotometry method
(Hirasa and Takemasa 1998). High-performance thin-layer chro-
matography (HPTLC) method has also been used for detection of
piperine in herbal formulations (Vyas and others 2013). HPTLC
is an enhanced form of TLC intended to increase the resolution
of the compounds to be separated and can provide more accurate
measurements. However, it is not as common as HPLC for detec-
tion of piperine. Analytical methods for the detection of piperine
are summarized in Table 3.
Enhancement of Bioavailability of Piperine
More than 40% of the identified herbal compounds are water-
insoluble (Merisko-Liversidge and Liversidge 2008). Piperine, as a
member of the lipid family, is also only sparingly soluble in water
and has a high lipid/water partition coefficient of 179.33 (log
P = 2.25; Kumoro and others 2009). Despite several astounding
therapeutic properties of piperine, its low solubility in water and
poor dissolution has limited its clinical efficacy. The hydrophobic
nature of piperine and its poor aqueous solubility is presumably
the main reason for the poor bioavailability of piperine. This is the
major hurdle for its development as a drug from lab to clinic, as the
low aqueous solubility of piperine poses a rate-limiting step in its
absorption process. This calls for new formulations to enhance its
aqueous solubility and thus make piperine more bioavailable. So
far, very little has been done in this regard and the reports dealing
with formulations of piperine for medicinal applications are very
scare in the literature; nanoformulations and encapsulations in
lipid bodies are among the approaches recently used to enhance
the bioavailability of piperine.
Nanoparticles
Drug delivery systems based on nanoparticles have become of
great interest in recent years. Nanoparticles have been used as phys-
ical drug carriers to change and improve the pharmacokinetic and
pharmacodynamic properties of different types of drug molecules
(Mohanraj and Chen 2007). These nanoparticles regularly have
particle sizes less than 1 μm and can improve oral bioavailability of
natural products. These systems release a certain amount of a drug
in specific locations, thereby they affect the pharmacokinetics and
drug distribution in the body (Merisko-Liversidge and Liversidge

C 2016 Institute of Food Technologists®
 
Table 2–A summary of implemented extraction methodologies for the isolation of the bio-active compound piperine from pepper corn.

Extraction Variety of Extraction yield Extraction

C 2016 Institute of Food Technologists®

method pepper (w/w) time Advantages Drawbacks Ref.
Piperine isolation from pepper . . .

DBSA Piper nigrum 3.90% ± 0.10% 12 ±1 h Simple, easy to operate High solvent consumption, Subramanian and others (2011)
long extraction time
Hydrotropic Piper nigrum 90% to 96% a 2h Few purification steps, little or Raman and Gaikar (2002a)
solubilization no foaming unlike the
surfactant solutions.
SFE Piper nigrum 6.7% to 7.6% 2 to 5 h Fast, selective, efficient, and High investment cost because Kurzhals and Hubert (1980),
clean method of the need to make Sankar (1989), Sovová and
equipment resistant to others (1995)
Piper nigrum 1.23% ± 0.05% 2.25 h high pressures Dutta and Bhattacharjee (2015)
(batch)
1.36% ± 0.02%
(continuous)
Enzyme-assisted SFE Piper nigrum 1.36% ± 0.04% 2.25 h Dutta and Bhattacharjee (2015)
(batch)
1.45% ± 0.04%
(continuous)
UAE Piper longum 0.58% 18 min Short extraction time, higher Requires filtering step and Rathod and Rathod (2014)
extraction yield and more small particle size of sample
comfortable control of
process parameters in
comparison to Soxhlet
White pepper 1.96% 18 min method Cao and others (2009)
ILUAE White pepper 3.577% 30 min High extraction efficiency, Cao and others (2009)
short extraction time,
environmentally friendly
nature
MAE Piper nigrum 94% (extraction 2 min Selective extraction, high Need to wait to cool down and Raman and Gaikar (2002b)
efficiency) extraction yield, short requires more filtration
extraction time steps than UAE
DBSA, double-bypass Soxhlet apparatus; SFE, supercritical fluid extraction; UAE, ultrasound-assisted extraction; ILUAE, ionic liquid-ultrasound-assisted extraction; MAE, microwave-assisted extraction.
a Recovery percentage, as declared by Raman and Gaikar (2002a); the authors of this work consider it as extraction efficiency.

Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 11
 Table 3–Analytical methods for the detection of piperine.
Piperine isolation from pepper . . .

Method Procedure Reference

Colorimetric Nitric acid method Piperine in concentrated nitric acid forms an unstable yellow color. When an alkali solution and Graham (1965c)
thiourea are added to it the color changes to red and becomes stable with maximum
absorption at 490 nm.
Komarowsky reaction When piperine is heated with the reagent containing para-hydroxybenzaldehyde, thiourea, Graham (1965a)
and concentrated sulfuric acid, a purple color solution with maximum absorption at 570 nm
forms.
Phosphoric acid method When piperine is heated in 85% phosphoric acid at 100 °C for 8 min, a stable bluish green Graham (1965b)
color solution with maximum absorption at 635 nm forms.
Column Detector Temperature
GC Capillary apolar column BP1 FID Injector 300 °C, detector 300 °C Noyer and others (1999)
UV spectropho- Piperine is dissolved in ethanol or methanol and the Singh and others (2011),

12 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
tometry absorbance is measured at 343 nm. Vishvnath and Jain (2011)
UV
HPLC Column Mobile phase wavelength Flow rate
C18 Acetonitrile: water (90:10) 343 nm 1.5 mL/min Rathod and Rathod
(2014)
C18 Dichloromethane: methanol (100:4) 343 nm 0.6 mL/min Raman and Gaikar
(2002a)
C18 Acetonitrile: water: acetic acid 340 nm 1 mL/min Upadhyay and others
(60:39.5:0.5) (2013)
HPTLC Toluene: ethyl acetate (70:30,v/v) 343 nm Raman and Gaikar
(2002b)
Benzene: ethyl acetate: diethyl ether 343 nm Vyas and others (2013)
(60:30:10)
GC, gas chromatography; HPLC, high-performance liquid chromatography; HPTLC, high-performance thin-layer chromatography.


C 2016 Institute of Food Technologists®
Piperine isolation from pepper . . .
2008). These structures can be considered as very effective drug
delivery system because they have advantages, including slow and
controlled drug release, protection of drug molecules, having par-
ticle size smaller than the cell, ability to cross biological barriers to
deliver drugs to the target, prolonging the retention time of a drug
in the bloodstream, targeted drug delivery, and biocompatibility
that enhances the efficacy of a drug (Benita 2005). Nanoparticles
as drug carriers also offer several considerable advantages over the
conventional drug delivery systems, such as high stability, high ca-
pacity for drug-carrying, controlled release of drug, and the ability
to deliver both hydrophilic and hydrophobic drug molecules (Pal
and others 2011).
There are many different methods for the preparation of a vari-
ety of nanoparticles, and some of them have been used for making
piperine nanoparticles. Pachauri and others (2015) worked on a
preparation of piperine-loaded poly (ethylene glycol)-poly (lactic-
co-glycolic acid) nanoparticles, denoted as PEG-PLGA, with the
solvent evaporation method, and they investigated its targeted de-
livery for adjuvant breast cancer chemotherapy. The average size
of synthesized nanoparticles was 132 nm. They reported that the
nanoparticles were stable in aqueous solution; and, in addition,
due to a decrease in metabolic rate, these nanoparticles could in-
crease the bioavailability of piperine and reduce its toxicity, if it
was used at higher concentrations. The synthesized nanoparticles
could preserve sustained release and targeted delivery of piperine
to the tumor site and they showed antitumor activity against dif-
ferent cancer cells; they also increased therapeutic capabilities of
other anticancer drugs. In another work, Jain and others (2016)
prepared a biodegradable polymeric system containing piperine
for cancer treatment. They prepared 30 wt% piperine-loaded
poly[Ɛ-caprolactone]/gelatin (PCL/GEL) nanofibers with diame-
ters between 300 and 400 nm and released. This piperine-loaded
PCL/GEL nanofiber that could release 50% of piperine within
3 d, showed in vitro anticancer activity against HeLa and MCF-7
cancer cells. Elnaggar and others (2015) prepared piperine-loaded
chitosan nanoparticles for brain-targeted therapy in Alzheimer’s
disease. Authors used encapsulation of piperine as an approach
to cover its pungency and to prevent the destruction of piperine
and also to reduce its irritability. They made spherical polymeric
nanoparticles with a particle size of 248.5 nm and an entrap-
ment efficiency of 81.7%. These nanoparticles not only improved
the cognitive effects as well as standard drugs, but also had the
additional advantage of a dual mechanism (inhibition of acetyl-
cholinesterase and antioxidant effect). They also reported that
piperine nanoparticles did not have any toxicity in the brain. In
another attempt, Yusuf and others (2013) produced polysorbate-
80 coated piperine solid-lipid nanoparticles (PS-80-PIP-SLN) by
emulsification-solvent diffusion method as a brain-targeted drug
for treatment of Alzheimer’s disease. The size of nanoparticles was
312 ± 5.1 nm and piperine entrapment capacity of the PS-80-PIP-
SLN was 68.2%. Results showed that the prepared PS-80-PIP-
SLN effectively targeted the brain, even at low dose of 2 mg/kg
bodyweight. It increased acetylcholinesterase value, decreased the
superoxide dismutase value and immobility, and showed better
results compared to chemical drug, Donepezil, which is used in
treatment of Alzheimer’s. Results also indicated that the impres-
sive therapeutic effects of PS-80-PIP-SLN in Alzheimer’s disease
treatment was through the reduction of the oxidative stress and
cholinergic degradation. In another investigation, Veerareddy and
Vobalaboina (2008) studied pharmacokinetics and tissue distri-
bution of piperine lipid nanospheres and observed that piperine
encapsulation in the lipid nanoparticles increased its mean resi-

C 2016 Institute of Food Technologists®
dence time in the blood stream. They also showed that piperine
lipid nanoparticles improved pharmacokinetic parameters com-
pared to a piperine solution.
Liposomes
Liposomes are spherical, closed structures that can be fabri-
cated from cholesterol and natural phospholipid bilayers and their
size ranges from 20 nm up to several micrometers. Liposomes
are biocompatible, biodegradable, and nontoxic artificial vesicles
and can be used as carriers for hydrophilic, hydrophobic, and am-
phiphilic drugs. Water-soluble drugs can be surrounded by the
aqueous center of the liposome, although lipid-soluble drugs are
inserted into the lipid bilayer of the liposome (Maherani and oth-
ers 2011; Akbarzadeh and others 2013). Pentak (2016) prepared
piperine-encapsulated nanosize liposomes by a modified reverse-
phase evaporation method. The size of particles was 100 to 20 nm
and the highest entrapment efficiency of nanoparticles was 90.5%;
the highest release percentage of piperine was observed in the 1st 3
h of incubation. Priprem and others (2011) worked on antidepres-
sant and cognitive effects of piperine-encapsulated liposomes. The
pungent odor of piperine was covered with the liposomal encap-
sulation and addition of polymers. The particle size of piperine
liposomes was less than 100 nm and the entrapment efficiency
of piperine was about 60%. Piperine could be delivered to the
brain when administered intranasally. Faster delivery rate of in-
tranasal piperine liposomes to the hippocampus with a higher
extent, compared to the oral dose, was observed. The intranasal
delivery of piperine from the liposomes could decrease the dose
of piperine intake, whereas its antidepressant and cognitive effects
were similar to those of oral dosage.
Self-emulsifying drug delivery systems
Lipid-based formulations for improving the bioavailability and
solubility of lipophilic drugs have attracted much attention re-
cently. Self-emulsifying drug delivery systems (SEDDSs) are
isotropic mixtures of natural or artificial oils and solid or liquid
surfactants which can have co-solvents (Gursoy and Benita 2004).
Shao and others (2015) used a SEDDS formulation for improving
the solubility and bioavailability of piperine. Ethyl oleate, Tran-
scutol P, and Tween 80 were used as the oil phase, co-surfactant,
and surfactant, respectively, because they had the highest piperine
solubility. The average piperine emulsion droplet size was 89.82
± 2.16 nm. The relative bioavailability of the developed SEDDS
formulation after oral administration improved by 625.7% as com-
pared to the self-prepared capsules. Also, the release rate of piperine
in SEDDS was significantly higher than that of the self-prepared
capsules. Spontaneous formation of emulsion and small droplet
size could be the reason for the quick release of piperine from the
SEDDS formulation. In intestinal absorption studies, the perme-
ability of SEDDS formulation was greater than that of the control
reference.
Microspheres
Microspheres are one of the most efficient approaches for sus-
tained and controlled drug delivery to a specific site. They are free-
flowing spherical-shape powders made of proteins and biodegrad-
able or nonbiodegradable polymers, and their particle size ranges
from 1 tp 1000 μm (Rastogi and others 2016). Adhesive, floating,
magnetic, and radioactive microspheres are kinds of microspheres
that are used in drug delivery systems. In the mucoadhesive type,
drug sticks to the mucosal membrane such as buccal, rectal, ocular,
and nasal mucosa, using water-soluble polymers which have a
Vol. 00, 2016 r Comprehensive Reviews in Food Science and Food Safety 13
Piperine isolation from pepper . . .
sticking property. In the floating type, the bulk density of mi-
crospheres is lower than that of gastric fluid which causes it to
remain floating in the stomach. Once gastric-emptying occurs,
the particles will distribute over a wide area of absorption sites
which increases the chance of drug release. In this system the rate
of drug release is slow and prolonged therapeutic effect is attained,
which decreases dosing repetition (Soppimath and others 2001;
Rastogi and others 2016). Boddupalli and others (2012) prepared
both floating and mucoadhesive gastro-retentive piperine micro-
spheres using the solvent evaporation method and investigated
their hepatoprotective and antiulcer activities. The particle size
obtained for floating and mucoadhesive microspheres was 114.5 ±
1.05 and 354.4 ± 5.25 μm, respectively. The results demonstrated
that floating piperine microspheres had better protection when
used against gastric ulcers compared to other formulations. They
showed superior hepatoprotective activity over mucoadhesive mi-
crospheres, possibly because of their lower particle size. The 2
piperine microspheres developed had greater hepatoprotective and
antiulcer activities compared to free piperine.
Hot melt extrusion
Hot melt extrusion (HME) is a simple and cost-effective ap-
proach of solid dispersion, which has gained attentions in the
pharmaceutical industry in order to enhance the bioavailability of
drugs, especially those with low solubility in water. HME improves
the drug bioavailability by dissolving or dispersing the drug in
melted polymer. Different kinds of polymers can be applied for the
solubility enhancement (Ridhurkar and others 2016). Ashour and
others (2016) used HME to increase solubility, permeability, and
R
oral absorption of piperine. Three different polymers: Soluplus ,
R
polyvinylpyrrolidone-co-vinylacetate 64 (Kollidon VA 64), and
R
Eudragit EPO, were used to investigate the effect of various
kinds of polymers on enhancing the solubility of piperine. Re-
sults showed 160- and 45-fold improvement in water solubility
of 10% and 20% piperine/Soluplus, respectively, and their drug
release profiles were remarkably increased. The possible reason
for good formulation stability is inhibition of drug recrystalliza-
tion and maintaining the drug in its amorphous form due to
R
the formation of hydrogen bonds between Soluplus and piper-
ine. Furthermore, compared to the pure piperine, an increase
in piperine absorption of 10% (w/w) piperine/Soluplus extru-
date was observed in permeability studies. These results demon-
strate that HME can increase the bioavailability and absorption of
piperine.
Conclusion
Piperine, the major pungent principle of pepper, is an alka-
loid with a remarkably broad spectrum of therapeutic activities.
It has also been shown to enhance the bioavailability of nutri-
tional and botanical compounds. In spite of the amazing ther-
apeutic properties of piperine, its pharmaceutical activities have
been limited due to its low aqueous solubility. The low solubility
of this bio-active compound in aqueous environments emphasizes
the development of new approaches which could contribute to
sustainable extraction of natural products, meanwhile offering en-
vironmentally benign and energy-efficient methods. Supercritical
CO2 extraction, ultrasound and microwave-assisted extractions as
well as IL and enzyme-assisted extractions, with some strengths and
advantages over traditional methods, are among the advanced tech-
niques implemented for piperine extraction; though each method
might have its own weaknesses and limitations. Although some
14 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2016
investigations have been undertaken for extracting piperine from
pepper and its purification, which is required for biomedical appli-
cations, little has been done for the direct use of extracted piperine
for therapeutic purposes. The low aqueous solubility of piperine is
the major barrier for its lab-to-clinic development as a drug. Even
though some attempts have been made to improve the bioavail-
ability of piperine through nanoformulations and encapsulation in
lipid bodies, it has a long way to go before it can be exploited as a
drug. The preliminary results reported for medicinal applications
of nanoformulated piperine are encouraging and, due to recent
advancements in the area of biotechnology, a bright prospect for
future therapeutic exploitation of piperine can be expected.
Authors’ Contributions
Author Gorgani collected the necessary information, Moham-
madi structured and drafted the manuscript, Najafpour and Nikzad
were involved in critically revising the work; all authors approved
the final version to be submitted for publication.
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