*Review* **Safety Aspects of the Use of Isolated Piperine Ingested as a Bolus**

**Rainer Ziegenhagen, Katharina Heimberg, Alfonso Lampen and Karen Ildico Hirsch-Ernst \***

German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, 10589 Berlin, Germany; rainer.ziegenhagen@bfr.bund.de (R.Z.); Katharina.Heimberg@bfr.bund.de (K.H.); Alfonso.Lampen@bfr.bund.de (A.L.)

**\*** Correspondence: Karen-Ildico.Hirsch-Ernst@bfr.bund.de

**Abstract:** Piperine is a natural ingredient of *Piper nigrum* (black pepper) and some other *Piper* species. Compared to the use of pepper for food seasoning, piperine is used in food supplements in an isolated, concentrated form and ingested as a bolus. The present review focuses on the assessment of the possible critical health effects regarding the use of isolated piperine as a single ingredient in food supplements. In human and animal studies with single or short-term bolus application of isolated piperine, interactions with several drugs, in most cases resulting in increased drug bioavailability, were observed. Depending on the drug and extent of the interaction, such interactions may carry the risk of unintended deleteriously increased or adverse drug effects. Animal studies with higher daily piperine bolus doses than in human interaction studies provide indications of disturbance of spermatogenesis and of maternal reproductive and embryotoxic effects. Although the available human studies rarely reported effects that were regarded as being adverse, their suitability for detailed risk assessment is limited due to an insufficient focus on safety parameters apart from drug interactions, as well as due to the lack of investigation of the potentially adverse effects observed in animal studies and/or combined administration of piperine with other substances. Taken together, it appears advisable to consider the potential health risks related to intake of isolated piperine in bolus form, e.g., when using certain food supplements.

**Keywords:** piperine; food safety; drug interaction; reproductive toxicity; bolus administration

## **1. Introduction**

The alkaloid piperine ((E,E)-piperine; IUPAC-name: (2E,4E)-5-(2H-1,3-benzodioxol-5 yl)-1-(piperidin-1-yl)penta-2,4-dien-1-one; CAS-No.: 94-62-2; FEMA-No: 2909; molecular formula: C17H19NO3; molecular weight: 285.34 g/mol) is a natural ingredient of *Piper nigrum,* Piper longum and some other *Piper* species, as well as of *Aframomum melegueta* K. Schum. (Grains of Paradise) [1–7]. The alkaloid is the main compound imparting the pungent flavour to fruits of *Piper nigrum* and *Piper longum*. *Piper nigrum* fruits are used to produce black, white and green pepper. Black peppercorns are produced from whole, dried, full-grown, not yet fully ripe fruits, while white peppercorns are produced from dried, ripe fruits after removal of the outer layer [8,9]. Green peppercorns are obtained from unripe fruits subjected to processing methods by which the green colour is maintained.

The occurrence of piperine in the European/Western diet primarily results from use of pepper for food seasoning, but also from the use of the substance in isolated form for spicing/flavouring purposes, e.g., in beverages and spirits [2]. The substance can occur in four stereoisomeric forms: (E,E)-piperine (= piperine), (Z,E)-piperine (= isopiperine), (E,Z)-piperine (= isochavicine) and (Z,Z)-piperine (= chavicine). In black and white pepper, (E,E)-piperine constitutes by far the main and most pungent isomer. The other three isomers seem to be formed primarily via light-induced or enzymatic isomerization [10].

**Citation:** Ziegenhagen, R.; Heimberg, K.; Lampen, A.; Hirsch-Ernst, K.I. Safety Aspects of the Use of Isolated Piperine Ingested as a Bolus. *Foods* **2021**, *10*, 2121. https://doi.org/10.3390/ foods10092121

Academic Editor: Dirk W. Lachenmeier

Received: 13 August 2021 Accepted: 6 September 2021 Published: 8 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Primarily in in vitro and in animal studies, as well as in some human studies, piperine has been shown to be a biologically versatile compound that can interact with a variety of chemically and functionally diverse biomolecular targets, such as enzymes, membrane transporters, receptors or other biomolecules. For example, piperine may provide protection against forms of oxidative damage and improve the activities of compromised anti-oxidative defence mechanisms (e.g., related to superoxide dismutase or catalase), but depending on the study settings, may also decrease the anti-oxidative defence mechanisms and among others, piperine further displays the potential to influence the activity of drugmetabolizing enzymes, including enzymes involved in phase I (cytochrome P-450-enzymes) and phase II metabolism (e.g., UDP-glucuronosyltransferases), to interact with cellular drug transporters (e.g., P-glycoprotein) or to modulate the cellular targets (monoamine oxidase) associated with neurodegenerative diseases [1,11–18].

Currently, attention is largely focused on the potential of piperine to influence the bioavailability of certain drugs via interaction with drug-metabolizing enzymes and/or inhibition of drug transporters or efflux pumps, thereby in many cases increasing the drug bioavailability and efficacy. In addition, inclusion of piperine into drug-loaded nanoparticles or lipospheres is being investigated as a means of increasing the effectiveness of advanced drug delivery systems [19,20].

In food supplements, piperine (primarily in the form of highly piperine-enriched pepper extracts, frequently with a piperine content in the range of ≥ 95%) is often used and promoted, among others, as a bio-enhancer to increase the bioavailability of other ingredients contained in these food supplements. Based on the use of piperine in various food supplements, its multi-facetted biological activities, and on the differences regarding the pattern of piperine intake when comparing the use of food supplements to the use of pepper for food seasoning, a closer look into the safety aspects of the use of piperine as an ingredient of food supplements appears to be warranted.

This review focuses on the possible critical health effects regarding the use of isolated piperine as a single ingredient in food supplements (i.e., without the addition of other bioactive substances). The use of isolated piperine as a flavouring agent is outside the scope of this review. In the context of the present review, the focus was laid on adult persons; thus, children and adolescents were not considered. To this end, a literature search was performed in the scientific databases Pubmed and Embase, with the last update performed in February 2021. To initially retrieve a broad spectrum of references, the search term "piperine" was used, without combination with other search terms. To further identify the relevant scientific publications that are within the scope of the present review, the abstracts of the retrieved references were screened to facilitate the selection of a subset of publications that were subsequently subjected to further scrutiny of the full texts. In addition, reference lists of the identified relevant publications as well as websites of acknowledged scientific bodies or national authorities were checked.

#### **2. Occurrence and Exposure**

#### *2.1. Occurrence*

*Piper nigrum* is the main source of piperine in European/Western cuisine. Other potential sources are foods flavoured with piperine in isolated form or foods flavoured with other *Piper* species (e.g., *Piper longum* and *Piper retrofractum* Vahl)or with the spice "grains of paradise" (*Aframomum melegueta*) [2]. Regarding the piperine content of black pepper, ranges of 2–7% [21], 2–9% [13] or 4–6% with contents up to 10% [2] have been reported. For *Piper longum*, piperine contents of 1.2–5% [13,22–24], and for *Piper retrofractum* Vahl contents of 3.1–4.5% have been indicated [13,23]. In an investigation of four commercial brands of pure ground black pepper with high piperine contents (10–11%), E,E-piperine was the most abundant (≥99% of detected piperine isomers) and Z,Z-piperine (= chavicine) the least abundant (≤0.07%) piperine isomere [10].

During storage of ground black, white and green pepper at 4 ◦C for 6 months, a decrease in piperine content of about 12–30% was observed [25]. Different findings were made regarding heat treatment of pepper, ranging from mild piperine losses of 4–12.5% during cooking in an open pan (30 min) or pressure cooking (20 min) [26] to losses of approximately 28% during cooking (20 min) or about 34% during pressure cooking (10 min) [27].

#### *2.2. Exposure*

In 2007, by extrapolation from limited consumption data, the Australian Therapeutic Goods Administration (TGA) estimated that the piperine intake in New Zealand was about 25 mg per person per day, in the USA approximately 60 mg and in India approximately 120 mg per person per day [28]. According to another source, which was based on annual US import data of black pepper, with an estimated average per capita intake of approximately 0.7 g pepper/day, a corresponding per capita piperine intake of 14–54 mg/day was calculated for the US population [29].

In an exposure estimation performed by the German Federal Institute for Risk Assessment (BfR) in 2018, which was based on food consumption data from the National Consumption Survey II of the Max Rubner-Institute (2008) [30], including approximately 20,000 individuals, a mean per capita pepper intake of the male German population (14–80 years) of 0.6 g/day was estimated, with an estimated per capita intake at the 95th intake percentile of 1.6 g pepper/day. Assuming an average piperine content in pepper of 4–6%, this would correspond to an estimated mean per capita intake by the male population of 24–36 mg piperine/day and an estimated intake at the 95th percentile of 64–96 mg piperine/day. Regarding this estimation, it is noted on the one hand that information on the consumption of herbs/spices is generally subject to greater uncertainty as their consumption is often not documented, and an underestimation of the amount consumed can therefore be assumed. On the other hand, it should be borne in mind that this intake estimation does not take into account possible piperine losses caused by storage or food preparation.

In India, a consumption survey conducted from December 2006 to July 2008 in three regions recorded the median monthly per capita intakes of black pepper of 3–18.5 g (0.1–0.62 g/day) and in the 90th percentile of 16.7–41.7 g (0.56–1.39 g/day) [31]

Taken together, the available estimations of daily piperine intake resulting from the use of pepper in food preparation are afflicted with considerable scientific uncertainty. It should also be kept in mind that when using pepper for food seasoning, the piperine intake occurs in conjunction with all other pepper constituents and with different degrees of comminution of the peppercorn, potentially bringing about matrix effects influencing the bioavailability or pharmacodynamic effects of piperine ingested in this way, which may differ from the intake of piperine as an isolated substance.

In its assessment of isolated piperine and several aliphatic and arylalkyl amines and amides as flavouring agents, the European Food Safety Authority EFSA (2015) reported an estimated European per capita intake of 6.2 μg piperine/day for the use of isolated piperine as a flavouring substance based on the EU Maximised Survey-derived Daily Intake (MSDI) method (see also below). However, EFSA noted in this assessment that the use levels were needed for some of the abovementioned flavouring substances, including piperine, to calculate the Modified Theoretical Added Maximum Daily Intakes (mTAMDIs) in order to identify those flavouring substances that required a more refined exposure assessment and to finalise the evaluation [32].

In food supplements, piperine is usually used in combination with other ingredients to increase their bioavailability, and commonly its addition occurs via highly piperineenriched black pepper extracts (piperine content frequently in the range of ≥ 95%). Therefore, black pepper extract is often mentioned on the ingredient list of food supplements and the piperine content is only indicated in second place. The piperine content of food supplements is frequently in the range of 5–30 mg per daily dose, with single products reaching dosages of 40 or up to about 50–100 mg per daily dose, but the market may be subject to change.

The piperine content of the highly piperine-enriched black pepper extracts (frequently in the range of ≥95%) is very similar to the piperine content of chemically defined piperine used as an isolated flavouring substance (piperine content ≥97% [33]) or to the piperine content of the substance used in scientific investigations, for which it was procured as a chemical from chemical companies (usually ≥97%).

#### **3. Kinetics and Metabolism**

In animal studies conducted by Bhat and Chandrasekhara [34,35] and Suresh and Srinivasan [36], with rats receiving an oral dose of 170 mg piperine/kg body weight (bw), only about 3–4% of the dose was detected in faeces in unchanged form over a period of 4 or 5 days, respectively, and it was concluded that 96–97% of the administered piperine dosage was absorbed [34,36]. In an accompanying investigation with everted sacs of rat intestines, only piperine was detected in serosal fluid and intestinal tissue, which led to the conclusion that piperine did not undergo any metabolic change during absorption [34]. However, in both the abovementioned animal studies, only small portions of the administered oral dose could be detected in serum and investigated tissues. In the more recent study of Suresh and Srinivasan (2010), maximum levels were reached 6 h after oral administration of a piperine dose of 170 mg/kg bw, with approximately 38.8 μmol piperine/L in serum and 0.39% of the administered piperine dose in liver, 0.37% in kidney and about 9.7% in the flushed intestine [36]. In both studies, no piperine was detectable in urine [34,36], but Bhat and Chandrasekhara detected piperine metabolites, i.e., piperonylic acid, piperonyl alcohol, piperonal and vanillic acid, and their conjugates, in urine, which in their free forms represented about 15.5% of the administered dose (measured within 96 h after piperine administration) [35]. The latter authors assumed that most of the administered piperine was absorbed and that it was not transformed during intestinal absorption but was probably later metabolized rapidly by other tissues [34].

In a more recent study in rats, the bioavailability of an oral dose of 3.5 mg piperine/kg bw was calculated to be about 25% by comparing plasma AUC values following oral and i.v. administration [37]. Regarding piperine metabolites, Gao et al. (2017) identified 12 metabolites in rat plasma, bile, urine and faeces, with 10 piperine metabolites occurring both in plasma and urine. The metabolites were grouped into metabolites resulting from methylenedioxycyclic ring-opening, from methylenedioxycyclic ring-oxidation and from piperidine ring-cleavage [38]. Shang et al. (2017) even detected and tentatively characterized 148 piperine metabolites in rat plasma, urine and faeces after oral administration of 250 mg piperine/kg bw. Piperine mainly underwent hydrogenation, dehydrogenation, hydroxylation, glucuronide conjugation, sulphate conjugation, ring cleavage and their composite reactions. However, information on plasma or urine levels of the detected piperine metabolites is not available from this study [39]. In laying hens receiving piperine–enriched feed (80 mg/kg feed), significant proportions of piperine isomers were observed in egg yolks (3.0 μg piperine, 0.7 μg chavicine, 2.9 μg isopiperine and 5.3 μg isochavicine per g egg yolk), indicating that piperine metabolism can also comprise substance isomerization [40].

Information on piperine serum or plasma levels observed in rats after oral administration is not uniform. With oral doses of 3.5, 20, 35 or 250 mg piperine/kg bw, corresponding plasma Cmax values of approximately 0.45, 3.4, 5.4–6.0 or 12.7 μmol piperine/L were observed in different studies [37,41–43]. However, other studies observed higher Cmax values with approximately 9.9 μmol/L after an oral dose of 20 mg piperine/kg bw [44] or levels of approximately 28–39 μmol/L after a dose of 170 mg piperine/kg bw [34,36]. Plasma protein binding was about 98% in rats receiving an oral dose of 35 mg piperine/kg bw [42]. Furthermore, piperine was shown to efficiently penetrate and homogeneously distribute into the brain of rats after oral piperine doses (35 mg/kg bw), leading to comparable AUC(0-∞)-values in brain and plasma with a brain–plasma AUC ratio of 0.95. However, based on the AUC(0-<sup>∞</sup>) values, the piperine level in cerebrospinal fluid was around 50 times lower than in brain or plasma [42].

In humans, information on kinetics and metabolism of oral piperine doses are sparse. In an investigation with two individuals receiving a single oral dose of 50 mg piperine (approximately 0.71–0.83 mg/kg bw, assuming a body weight of 60–70 kg), the plasma peak concentrations reached 2.7–3.3 μmol/L (Tmax = 1–3 h) [45].

In human urine, the piperine metabolites 5-(3-4-dihydroxphenyl)valeric acid piperidide (which was excreted as sulphate) and its derivate hydroxylated in position 4 of the piperidine ring, 5-(3-4-dihydroxphenyl)valeric acid-4-hydroxypiperidide, were observed one or two days after oral administration of piperine (25 mg) or a high dose of pepper, respectively. Interestingly, these two urine metabolites could not be detected in 2 out of 14 investigated individuals who instead excreted 5-(3-4-dihydroxphenyl)-2-4-pentadienoic acid piperidide, providing first indications for individual differences in human piperine metabolism [46]. In rat urine, all three metabolites could be detected [38]. In an in vitro study comparing the hepatic piperine metabolism in mouse, rat, dog and human hepatocytes, the predominant metabolic pathways included formation of a catechol derivate for all species; however, the metabolic pathways displayed species-specific differences in terms of types and quantities of metabolites [47].

## **4. Safety Aspects**

#### *4.1. Information Based on Evaluations by Scientific Bodies and National Authorities*

The European Food Safety Authority (EFSA) has evaluated the use of piperine as a flavouring substance. In its evaluations, EFSA (2008; 2011; 2015) disagreed with a No Observed Effect Level (NOEL) of 20 mg piperine/kg bw/day that had previously been identified by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (2006), due to the shortcomings of the underlying animal study (lack of histopathology, study duration) and in 2015 identified a No Observed Adverse Effect Level (NOAEL) of 5 mg piperine/kg bw/day, based on a newly available 90-day rat feeding study performed according to OECD guideline 408 (endpoint: dose-dependent increase in cholesterol level in male animals) [29,32,48–50] (see (2) in Section 4.2.2). In its final conclusion, EFSA (2015) agreed with the JECFA (2006) conclusion "no safety concern at estimated levels of intake as flavouring substance" based on the MSDI approach (estimated European per capita intake by the MSDI approach: 6.2 μg piperine/day) [32,50]. Currently, isolated piperine is approved as a flavouring agent in the European Union with no restrictions on use or maximum levels set in regulation (EC) No. 1334/2008. However, its use level may be self-limiting due to the pungent taste of piperine.

In 2007, the Australian Complementary Medicines Evaluation Committee (CMEC) evaluated the use of piperine as a component in herbal preparations for use in listed medicines. Due to the possible effects on the bioavailability of medicinal products (leading to increased bioavailability in most cases, see Section 4.2.4) and the risk of inadvertent interactions with medicinal products, the committee recommended a maximum daily dose limit of 10 mg/day for piperine (based on a person's body weight of 50 kg) when present as a component in herbal preparations for use in listed medicines [28].

The Canadian authority Health Canada (2019) has elaborated a monograph on the use of *Piper nigrum* (black pepper) as an ingredient in *Natural Health Products,* which also includes piperine isolated from the fruits of *Piper nigrum*. For adults (≥18 years), a daily dose of 250–420 mg for the unextracted powder of *Piper nigrum* fruits and a daily maximum dose of 14 mg for the use of piperine as an isolated substance in these products were established. For these products, a label statement is required that persons taking other medicines or natural health products should consult a healthcare practitioner/provider/professional or physician before use, as black pepper/piperine may alter their effectiveness. The same applies to pregnant or breastfeeding women. The monograph does not list any contraindications or known adverse reactions [51].

None of the evaluations described above mentioned the paternal reproductive toxicological effects observed in some animal studies (see (3) in Section 4.2.2).

In 2016, on request of the Norwegian Food Safety Authority, the Norwegian Scientific Committee for Food Safety (VKM) carried out a risk assessment of a daily dose of 1.5 mg piperine in food supplements. The panel applied the Margin of Exposure (MOE) approach in its assessment and used the NOAEL of 5 mg/kg bw/day identified by EFSA (2015), which was based on an animal study (endpoint: dose-dependent increase in cholesterol level in male animals) as the starting point for the MOE calculation. As the margin of exposure for all age groups considered was greater than 100, the panel concluded that this intake was unlikely to produce adverse effects in individuals aged 10 years or older [2].

#### *4.2. Potential Hazards*

#### 4.2.1. Genotoxicity

In its assessment of piperine as a flavouring agent, JECFA (2006) concluded regarding genotoxicity that piperine belongs to a group of aliphatic and aromatic amine and amide derivates for which negative results were reported in bacterial assays for reverse mutation and that piperine consistently gave negative results in a variety of in vivo studies [50,52–54]. EFSA agreed in its assessment with JECFA that the available studies on genotoxicity did not preclude the evaluation of piperine (and some other aliphatic and arylalkyl amines and amides) as a flavouring agent [32,48,49].

In a more recent study, piperine displayed negative results in an in vitro micronucleus test with Chinese hamster ovary cells in the presence or absence of metabolic activation and caused no increase in the numbers of micronucleated polychromatic erythrocytes in an in vivo micronucleus test in mice with all the tested doses (highest tested dose 574 mg/kg bw), leading to the conclusion that in this study piperine was not genotoxic [55].
