A Systematic review of B-caryophyllene and Piperine as Multitarget Phytochemicals: Integrated Phytochemical Profiling and Mechanistic Insights into their Biological Activities

Phytochemicals (medical plant extracts) will be a pillar both of traditional medicine and pharmacology discovery. With the world becoming more and more multi-factorial chronic disease-prone and antimicrobial resistance-induced and single-target synthetic drugs constrained, phytochemicals are an appealing paradigm of multi-target, pleiotropic action [1]. These are secondary metabolites, which have evolved as either tools of a plant defense strategy or signaling, and include a rich chemical perspective- polyphenols and alkaloids to terpenes and glycosides- with bioactive potential as diverse as possible. Contemporary therapeutics are exploiting these compounds not only as crude extract but as defined molecular structures where their exact mechanism of action, synergistic and scaffolding properties are studied and utilized in the design of novel drugs [2]. The importance of such transition to evidence-based molecular pharmacology cannot be overstated, as strict phytochemical analysis is essential to isolate, identify and purify bioactive components to convert traditional remedies into reproducible and mechanism-based interventions. Among these vast arrays of phytochemicals, two terpenes, β-caryophyllene and piperine, are of particular interest and complement to one another; β-caryophyllene is a bicyclic sesquiterpene that is common to the essential oils of many culinary and medicinal plants, such as black pepper (Piper nigrum), cloves (Syzygium aromaticum), cannabis (Cannabis sativa), and oregano (Origanum vulgare)[3-5]. It is unique in being the first known dietary cannabinoid, being a selective full agonist of the cannabinoid receptor type 2 (CB2), which is present majorly in the peripheral tissues and immune cells. This interaction with receptors, which is free of psychoactivity via the CB1 receptors, is the basis of its highly recorded anti-inflammatory, antioxidant and analgesic effects[6]. Being a volatile compound, its bioavailability is frequently a restrictive concern, and its interaction with absorption enhancing agents is therefore a strong subject of investigation [7]. On the other hand, the major bioactive alkaloid constituent of black and long pepper, and the most prominent example of this group, is called piperine; it has been recognized to have no individual receptor target, but has incredibly high pharmacokinetic modulation potential [8]. It is known to be a powerful bioavailability enhancer or bioenhancer of a great variety of other co-administered drugs and nutrients. Its action involves them being multifaceted, such as inhibition of drug-metabolizing enzymes, such as cytohemoglobin P450 and UDP-glucuronosyltransferase, suppression of P-glycoprotein efflux transporters, and stimulation of gastrointestinal amino acid transporters [9]. In addition to playing this facilitator role, piperine has inherent pharmacological properties, having been shown to have anti-inflammatory, hepatoprotective, and neuroprotective effects, which in many cases are mediated by its effect on transient receptor potential vanilloid (TRPV) channels and additional cellular signal pathways [10-13].

The argument behind the research of β-caryophyllene and piperine together is solid and multilevel. To begin with, the synergy between the phytochemical properties of black pepper is strong, due to the fact that the plant species naturally co-exist and co-purify together [14]. Second, and more fundamentally as a drug matter would suggest, is the postulate of a pharmacodynamic-pharmacokinetic (PK-PD) synergy. The poor aqueous solubility, rapid metabolism, and low systemic exposure of many lipophilic terpenes such as β-caryophyllene inherent to them might be directly overcome by bioenhancing agents such as piperine. This blend is not only likely to increase plasma concentration and tissue bioavailability of β-caryophyllene but also, it may have the capacity to increase the therapeutic effect [15-17]. Moreover, their pharmacological actions can be synergistic or antagonistic as the example of the anti-inflammatory effects of β-caryophyllene with NF-kB through CB2 stimulation and the ability of piperine to regulate inflammatory pathways such as NF-kB and COX-2. The study of this combination is no longer simply the study of individual compounds but itself is a more holistic systems pharmacology, which is more reminiscent of the poly-pharmacology of whole plant extracts [18].

Thus, the purpose, as well as the scope of this systematic review, is to systematically collect, critically evaluate, and synthesize the available scientific data on the phytochemical investigation, biological activities, and the interactive impact of β-caryophyllene and piperine. The scope includes preclinical in vitro and in vivo studies that describe their extraction, characterization, individual pharmacological actions and most importantly, a research describing their interaction [19]. The review will outline the molecular processes involved in their action, review the findings of synergistic interaction, and analyze the toxicological profile of the combination. The review is organized according to important research questions and hypotheses in order to direct this investigation [20-22].

2. METHODOLOGY: SYSTEMATIC REVIEW PROTOCOL

Overall extensive and planned search strategy was developed and implemented to find all the literature. The core electronic databases that were searched were PubMed/ MEDLINE, Scopus, Web of Science Core Collection, and EMBASE. A systematic combination of keywords and Medical Subject Headings (MeSH) terms of the core compounds was used in the search. The search keywords were beta-caryophyllene/ b-caryophyllene/ caryophyllene/ piperine/combined with (AND, OR) Boolean operators. The search focused solely on studies in English, and there was no time constraint on the date of publication, although it focused on recent analysis. All articles included and all the relevant reviews had their reference lists screened manually to ensure that no more sources not included in the database searches were found.

Eligibility Criteria (Inclusion and Exclusion Criteria)

3. PHYTOCHEMICAL PROFILING AND ANALYSIS

The demanding nature of bioactive compounds characterization is essential to consistent pharmacological studies, and the body of traditional plant-based knowledge is converted to proved scientific investigation [23]. The following section also outlines botanical origins, methods of extraction and analysis of β-caryophyllene and piperine which serve as a foundational component of the aroma and biological protection of the majority of plants of various families. It is mostly an ingredient in the essential oils of herbs and spices that are used as essential oils with food and as a result, it is mostly found as a frequent supplement in food. The most important commercial and research source is clove (Syzygium aromaticum) in which the essential oil is largely a significant fraction of β-caryophyllene (about 5-15%), and the oxidized derivative, caryophyllene oxide, and phenylproanoid eugenol are also commonly present. Clove oil is still used as a standard with regard to the distillation of high-purity b-caryophyllene [24]. Likewise, black pepper (Piper nigrum), which is also the source of piperine, also provides important amounts of β-caryophyllene in its volatile oil, which provides a natura combinatorial source. The Cannabaceae family, particularly hemp and some chemovars of Cannabis sativa have been of great interest to researchers due to their richness. Β-caryophyllene is a potent sesquiterpene in the terpene profile of the cannabis plant, which interacts with cannabinoids in an entourage effect, and has non-psychoactive therapeutic potential because of CB2 receptor activation. Oregano (Origanum vulgare), cinnamon (Cinnamomum spp.), hops (Humulus lupulus) and a range of balsamic resins of trees of the genus Copaifera (e.g., Copaifera langsdorffii) are other notable botanical sources of this compound, which also produce copaifera balsamic resins in abundance [25-27]. The difference in β-caryophyllene content between the sources of plant depends on the geographical origin, genotype, and plant part (bud, leaf, stem), harvesting conditions; therefore, the accurate botanical identification and phytochemical standardization are indispensable. The volatility and lipophilicity of β-caryophyllene are used to isolate the compound by taking advantage of the plant matrices. Traditional methods are characterized by hydrodistillation and steam distillation, in which plant substance is heated using boiling water or steam, which causes the essential oil that includes β-caryophyllene to evapour, after which the essential oil is then condensed and separated. Although these thermal methods are effective and scalable, it is sometimes up to the oxidation or hydrolysis of the terpene profile, or the degradation of heat-sensitive compounds. A frequent method is solvent extraction which involves the use of non-polar solvents such as hexane or dichloromethane and affords a crude extract that is extracted further to obtain b-caryophyllene [28].

Advanced extraction methods have been on the increase in the quest to achieve greater yields, selectivity and greener processes. A gold standard of terpene isolation is Supercritical Fluid Extraction (SFE) mainly in which carbon dioxide (CO₂) is the solvent. SFE uses solvating power by means of controlling temperature and pressure to achieve high solvating power selectively and tunably above the critical point of CO₂. It works at close to ambient temperatures, eliminating thermal degradation and does not leave behind a toxic residual solvent which forms a high-quality, "full-spectrum" extract that does not alter the native terpene profile including b-caryophyllene [29]. Other new innovations include Microwave-Assisted Hydrodistillation (MAHD) and Ultrasound-Assisted Extraction (UAE). In order to extract the essential oil, MAHD applies the microwave energy to heat the water inside the plant cells quickly and rupture them, which is more effective than traditional heating methods in extracting the essential oil, thus a shorter extraction time and less power is used [30]. UAE uses ultrasonic cavitation to rupture cell walls and improve the mass transfer of the target compound to the solvent. The extraction technique utilized also has a major effect on the purity and recovery of β-caryophyllene and also on the co-extraction of any other bioactive constituent, which may affect the resulting biological activity of the extract [31].

3.1. Sources and extraction methods for Piperine

In contrast to the widely spread b-caryophyllene, the distribution of the more specific alkaloid, piperine, is nearly limited to the genus Piper in the Piperaceae family. The best is the fruit of black pepper (Piper nigrum), which is of the most commercial spices in the world. The main component that makes black pepper pungent is called Piperile and it usually amounts to 5-9% of the dry weight of the pepper, however this can be variably changed with sources of pepper and processing (black, white or green pepper). The other prominent spice of the traditional medicine systems including Ayurveda, long pepper (Piper longum) is also a good source of piperine and associated bioactive alkaloids like piperlongumine [32]. Though there are other species of Piper which could also include piperile and analogs, P. nigrum and P. longum are the most common species to extract commercially and do research on the pharmacology because they have high alkaloid content and can be grown on the farm. Piperine is extracted and purified because it is an alkaloidal compound that is relatively stable. Soxhlet extraction is the most traditional and the most common laboratory process. Ground pepper powder is put in a thimble in this continuous procedure and repeatedly rinsed with a hot, non-polar or moderately polar solvent (which typically is ethanol, methanol, dichloromethane, or acetone). It is a good technique of exhaustive extraction but is time-consuming and consumes much solvent. A less efficient, but less complicated technique commonly applied to initial crude extraction is maceration with stirring (agitation) in solvents such as ethanol [33]. After removing the solvents in a low pressure process, the crude piperine extract left behind is a resinous substance with fats, oils and other piperamides. To do the analytical and pharmacological studies, the purification of the piperine in this crude extract is essential. Recrystallization is a widely used procedure. This is then dissolved in a warm appropriate solvent (e.g. ethanol or acetone), when cooled pure piperine crystals are obtained and impurities are left in solution. This can be repeated to attain a greater purity [34]. To achieve high purification or separation of complex mixtures then chromatographic methods are used. Piperine can be separated, by column chromatography upon silica gel, by means of gradient elution, with solvents of increased polarity (e.g. hexane to ethyl acetate) to separate it with other closely-related piperamides. Thin-layer Chromatography Thin-layer chromatography (TLC) or High-Performance Liquid chromatography (HPLC) are powerful methods of isolating milligram to gram quantities of high-quality piperine to be used in research [35]. A simple recrystallization versus the complicated chromatography will be dependent on the purity needed, and the properties of the co-extracting compounds of the starting material. The absolute recognition and the measurement of β-caryophyllene and piperine are unconditional requirements of quality control, standardization, and valid bioactivity research. The contemporary study of phytochemicals is based on the synergistic approach to the use of chromatographic and spectroscopic procedures. Chromatography splits up complex mixtures permitting the extraction and quantification of the individual components. The most superior method of analyzing volatile and semi volatile substances such as β-caryophyllene is the Gas Chromatography-Mass Spectrometry (GC-MS) [36]. In GC, the sample is vaporized and transported by an inert gas over a column which is coated and the components are separated according to their boiling points and attraction to the column coating. The compounds that are separated are then passed through a mass spectrometer that breaks them down into ions. The mass spectrum that is obtained is a molecular fingerprint. The retention time and mass spectral fragmentation pattern and β-caryophyllene is commonly recognized by comparing them to those of a known standard in known databases (e.g., NIST, Wiley). GC-MS too is quantitative and the concentration of b -caryophyllene in essential oils or extracts can be determined accurately [37].

Advanced extraction methods have been on the increase in the quest to achieve greater yields, selectivity and greener processes. A gold standard of terpene isolation is Supercritical Fluid Extraction (SFE) mainly in which carbon dioxide (CO2) is the solvent. SFE uses solvating power by means of controlling temperature and pressure to achieve high solvating power selectively and tunably above the critical point of CO2 [38-42]. It works at close to ambient temperatures, eliminating thermal degradation and does not leave behind a toxic residual solvent which forms a high-quality, "full-spectrum" extract that does not alter the native terpene profile including b-caryophyllene. Other new innovations include Microwave-Assisted Hydrodistillation (MAHD) and Ultrasound-Assisted Extraction (UAE). In order to extract the essential oil, MAHD applies the microwave energy to heat the water inside the plant cells quickly and rupture them, which is more effective than traditional heating methods in extracting the essential oil, thus a shorter extraction time and less power is used. UAE uses ultrasonic cavitation to rupture cell walls and improve the mass transfer of the target compound to the solvent. The extraction technique utilized also has a major effect on the purity and recovery of β-caryophyllene and also on the co-extraction of any other bioactive constituent, which may affect the resulting biological activity of the extract [43].

High-Performance Liquid Chromatography (HPLC) is preferred in the instance of compounds that are not volatile and which are thermally labile such as piperine. In this case, the sample is pumped through a column filled with fine particles (stationary phase) by a liquid solvent (mobile phase). Separation is done according to the difference interaction with the stationary phase. HPLC is normally combined with Ultraviolet (UV) or Photodiode Array (PDA) detection. Piperile is highly UV absorbing with typical maximal at 343 nm which can be quantified sensitively and selectively [44]. HPLC-MS is a combination of the separation ability of HPLC with the conclusive identification ability of mass spectrometry that offers even higher confidence in the analysis of the piperine and its metabolites in the complex biological samples.

The more sophisticated variant of TLC, High-performance Thin-layer Chromatography (HPTLC), is a useful and economical method of qualitative screening and semi-quantitative analysis. It enables the analysis of several samples and standards on a single plate at a time. Once in a solvent system, plates can be seen under UV light or reacted with certain reagents to form colored spots [45]. These bands can also be densitometrically scanned to quantify such compounds as the piperine and b-caryophyllene, which makes HPTLC a good method to fingerprint and determine the quality of plant materials and formulations. Whereas chromatography is used in the separation and quantification, spectroscopy is used in the elucidation of molecular structure. Structural elucidation of pure isolated compounds is unquestionably best done through Nuclear Magnetic Resonance (NMR) Spectroscopy. It gives information on the carbon-hydrogen structure of a molecule in finer details like the number of hydrogen atoms, their type, and connectivity and gives the carbon skeleton of the molecule through 13C NMR. In the case of piperine, NMR confirms the existence of the piperidine ring, methylenedioxy phenyl group and chain of conjugated diene. In the case of b-caryophyllene, it validates the complicated bicyclic framework that has the characteristic cyclobutane and nine-membered ring. The identity of a pure compound and confirmation of purity is the ultimate confirmation to identify that the NMR spectrum of an isolated compound compares to published data of a known standard. The Fourier-Transform Infrared (FT-IR) Spectroscopy is a technique used to quantify molecular bonds on the basis of their absorption of infrared light to give a spectrum indicating functional groups. It is used as a complementary method to NMR. The FT-IR spectrum of piperine has strong characteristic peaks in the amide carbonyl, (C=O stretch -1630-1680 cm -1), aromatic, and alkene C-H stretches and the methylenedioxy groups [46-49]. The spectrum of β-caryophyllene indicates the presence of alkene (=C-H stretch -1680 cm -1) C-H and none of the carbonyl and hydroxyl groups, agreeing with its hydrocarbon nature. FT-IR is fast and effective in the preliminary characterization of the compound and identification of significant functional group alterations.

4. PHARMACOKINETICS AND BIOAVAILABILITY

The therapeutic capability of any bioactive compound is essentially limited by their pharmacokinetic profile-absorption, distribution, metabolism and excretion (ADME) process in the body. The unique ADME properties of β-caryophyllene and piperine, and more importantly their interaction, is the key to rationalizing their combined application and opening up synergistic effects. Absorption, Distribution, Metabolism and Excretion (ADME) of β-caryophyllene β-caryophyllene is highly lipophilic sesquiterpene hydrocarbon, hence good oral absorption is facilitated by favourable passive diffusion through the biological membranes. Research points out that it is quickly absorbed through the gastrointestinal tracts of the body, with plasma concentrations being felt within minutes. Its high hydrophobicity however is also a major challenge hence poor solubility in aqueous system and poor solubility in gastrointestinal fluids which is a typical rate limiting step to many lipophilic drugs [50]. When ingested, β-caryophyllene is highly distributed in the tissues, and has a restricted ability to cross the blood-brain barrier, but instead, is accumulated in lipid-rich organs, which include liver and adipose tissue. It has a high and widespread metabolism which is hepatic and catalyzed by cytochrome P450 enzymes (especially CYP2C9, CYP2C19, and CYP3A4). The key metabolic routes imply oxidation that results in the production of β-caryophyllene oxide and several hydroxylated derivatives which are further conjugated with glucuronic acid to be excreted through the kidneys. This rapid hepatic first-pass metabolism and systemic clearance leads to a relatively short plasma half-life and low oral bioavailability in its native form which is commonly cited as a major reason behind its inability to sustain therapeutic effect when utilized alone [51]. 

4.1. Pharmacokinetic profile of Piperine

Piperine, being also lipophilic, shows a more complicated pharmacokinetic profile characterized by the ability of a strong modulator of drug disposition systems Fig.1. Piperile given orally is not efficiently absorbed, but its own bioavailability is moderate, and depends on its metabolism. It is wide spread in distribution. The actual pharmacokinetic importance of piperine is not in the real ADME parameters of this drug itself but in the fact that it has an immense inhibitory effect on the important enzymatic and transport systems [52]. Piperile is largely metabolized in the liver and the intestine but also acts as a phase I and phase II metabolism inhibitor of wide spectrum. More importantly, it has been identified to inhibit CYP enzymes (such as the 3A4 and 2C subfamilies) and, most significantly, glucuronidation enzymes, uridine 5'-diphospho-glucuronosyltransferase (UGT). In addition, it block the efflux pump of P-glycoprotein (P-gp) that comes about in the intestine, and other tissues, which returns taking drugs into the gut lumen and limits its exposure to the systemic tissues [53]. Mechanistic Insights Piperine is considered by its reputation as a bioenhancer because of its multi-pronged interference with the natural defense systems of the body against xenobiotics. Three main mechanistic domains of its action may be identified: pre-systemic, systemic and cellular. At the pre-systemic (intestinal) site, piperine raises intestinal epithelial permeability, which may be through membrane dynamics and P-pg inhibition and thus more of a co-administered drug enters the portal circulation. Its blockage of CYP and UGT hepatic enzymes liver decreases the first-pass metabolism of compounds which are substrates of those enzymes in the systemic (hepatic) domain. This reduces their metabolism into inactive metabolites, and therefore, a higher percentage of the parent drug gets to the systemic circulation [54]. At the cellular level, drugs may be inhibited in P-gp and other efflux transporters in different tissues to enhance intracellular concentration and decrease biliary or renal excretion. Also, amino acid transporters in the gut may be activated by piperine and increase blood flow to the gastrointestinal which also promotes absorption. It is this multi-target pharmacological braking effect on elimination pathways that contributes to greatly increasing bioavailability of an extensive range of compounds including anti-tuberculosis drug rifampicin as well as phytochemical curcumin [55].

Fig: 1 Pharmacokinetic profile of Piperine

4.2. Pharmacokinetic profile of β-caryophyllene

Studies Evidence Since the ADME limits of β-caryophyllene are known and the bioenhancing capability of piperine is broad, a combination of the two is a logical pharmacokinetic approach Fig.2. The preclinical evidence is quite strong in favor of this hypothesis [56]. Pharmacokinetic experiments performed in animal models which expose β-caryophyllene together with piperine also show that the systemic exposure of β-caryophyllene is dramatically enhanced. Among the important ones are marked escalation of the highest plasma focus (Cmax) and a marked escalation of the region under the plasma focus-time curve (AUC), a direct quantification of complete systemic exposure. As an example, a study revealed that oral bioavailability of β-caryophyllene can be increased many times using piperine [57]. The direct cause of this improvement is the mechanisms of piperine: blocking the CYP enzymes of oxidative metabolism of β-caryophyllene and the UGT enzymes of its glucuronidation, piperine decreases its metabolism. At the same time, the intestinal P-g inhibition can enhance net absorption. Not only does the outcome increase the plasma levels but also the mean residence time becomes longer, virtually increasing the therapeutic window of action. This pharmacokinetic interaction directly relates into enhanced and protracted pharmacological actions in disease models that make it possible to reduce and decrease dosing of β-caryophyllene to obtain the intended results with less and less dosing. This data is a key stone in the integrated application between these two phytochemicals to convert β-caryophyllene into a compound with a moderate bioavailability into one with a strong and consistent pharmacokinetics with the ability to deliver on its therapeutic potential [58].

Fig: 2 Pharmacokinetic profile of β-caryophyllene

5. BIOLOGICAL PROPERTIES AND PHARMACOLOGICAL ACTIVITIES

The potential to treat various conditions using β-caryophyllene and piperine is supported by their various and strong biological functions, which cut across key physiological processes of cellular defense up to systemic homeostasis. Critical synthesis of preclinical studies indicates that both pharmacological profiles are not only strong but can be complementary and this gives an interesting argument behind their combination [59]. This section defines the main biological characteristics of each compound, explaining their molecular responses and pointing at the evidence of emergent interactions to the synergies.

5.1. Anti-inflammatory and immunomodulatory effects

Chronic inflammation is an underlying pathological process of a multitude of diseases and both β-caryophyllene and piperine demonstrate pronounced anti-inflammatory and immunomodulatory effects by unique but possibly overlapping mechanisms, namely via the cannabinoid receptor type 2 (CB2) [60].  β-caryophyllene is an activator of CB2 which provokes a signaling cascade that suppressed the Nuclear Factor-kappa B (NF-kβ) pathway, one of the primary regulators of inflammation, potently. The result of this inhibition is the downstream inhibition of pro-inflammatory cytokines and mediators, such as Tumor Necrosis Factor-alpha (TNF-a), Interleukin-6 (IL-6), and Cyclooxygenase-2 (COX-2) which is an enzyme that produces prostaglandins.

 5.2. Antioxidant and cytoprotective activities

Being a free radical scavenger, β-caryophyllene is capable of counteracting the effects of ROS and, by doing so, protect cellular lipids, proteins, and DNA against oxidative stress. But a more important role can be as an indirect antioxidant by the stimulation of the Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Activation causes translocation of Nrf2 to the nucleus, which binds to the Antioxidant Response Element (ARE), which upregulates the expression of a collection of cytoprotective enzymes, such as heme oxygenase-1 (HO-1), NADPH quinone dehydrogenase 1 (NQO1), and glutathione S-transferase (GST) Fig.3.

Fig: 3 (a) Immunomodulatory Effects of β-Caryophyllene in Inflammation and Viral Entry Inhibition (b) Mechanism of Piperine- effects on  Allergic Reactions