CHEMICAL ANALYSIS OF (E)-β-CARYOPHYLLENE

The chemical composition of PipeNig® is characterized by the presence of sesquiterpene (E)-β-caryophyllene, a sequiterpene hydrocarbon (Figure 1).

La composizione chimica di PipeNig® è caratterizzata dalla presenza del (E)-β-cariofillene, un sequiterpene idrocarburico (Figura 1).

Pipenig-facts-1

Figure 1: Structure formula of (E)-β-caryophyllene

PipeNig® is analyzed by gas chromatography (GC) coupled to mass spectrometry (MS) for qualitative analysis of the compounds and by GC coupled to flame ionization detection (FID) for quantitative analysis. GC-FID quantitative analyses revealed a total percentage of 80% (E)-β-caryophyllene in the volatile fraction of PipeNig®-FL and 30% in PipeNig®-PWD. The precise quantification of (E)-β-caryophyllene and its standardization in PipeNig® allows the preparation and accurate dosage of products based on PipeNig®. For instance, in a recent pilot study (see below) we tested the effect of capsules contaning 100 mg PipeNig®-PWD, corresponding to 30 mg (E)-β-caryophyllene. Figure 2 shows a typical GC-MS profile of PipeNig®.

pipenig_fig-2_gc-ms

Figure 2: GC-MS chromatogram of PipeNig®

ANALGESIC PROPERTIES OF PIPENIG®: A PILOT STUDY

The endogenous cannabinoid system (ECS) plays an important role in the immune response to an infection. At present, two cannabinoid (CB) receptors are described: cannabinoid type 1 receptor (CB1) and cannabinoid type 2 receptor (CB2), both G-protein coupled receptors. CB2 receptor represents the peripheral CB, due to its expression on circulating immune cells. However, studies have also found CB2 expression in the brain, such as cerebellum and microglial cells. CB2 receptor is involved in the attenuation of inflammatory immune responses. CB2 receptor ligands have been shown to inhibit inflammation and edema formation, exhibit analgesic effects, play a protective role in hepatic ischemia-reperfusion injury, and prevent experimental colitis by reducing inflammation.

CB2 receptor pathway activation entails the suppression of cytokine release from immune cells and thereby dampening of the inflammatory response (immunosuppression). CB2 receptor-selective agonists that are devoid of the psychoactive side effects typically associated with CB1 receptor activation are potential candidates as a valid support of a range of different diseases. (E)-β-caryophyllene (BCP, Figure 1) is a sesquiterpene hydrocarbon that selectively binds to the CB2 receptor and is a functional CB2 agonist. BCP exerts toxicity at doses higher than 2000 mg/kg body weight and also has an anxiolytic-like and anti-depressant effects. The possibility that BCP may ameliorate mood disorders offers exciting prospects for future studies

A consistent number of in vitro and in vivo studies on BCP indicates that its biological effects include anti-inflammatory, antimicrobial, antioxidant and analgesic activities. These biological effects derive from the ability of BCP to activate the CB2 endocannabinoid receptor, thus behaving like an endo-cannabinoid. The specificity of BCP for the CB2 receptor, mainly expressed in peripheral tissues, and its inability to bind CB1 which is predominantly expressed at the level of the central nervous system, implies that its action is devoid of the known psychoactive effects associated with the activation of CB1 and suggests its potential use. Thus, BCP offers the possibility of obtaining a pain-killer and/or anti-inflammatory action as an interesting alternative to the use of Cannabis.
Recent findings have shown significant implications for clinical research and strongly supports the effectiveness of BCP as a novel molecule to target in the development of effective therapeutic agents for multiple sclerosis. In a pilot study performed by the Farmacia Centrale (Cambiano, Italy), a balanced sample of 31 volunteers (19 females and 12 males, with ages ranging from 36 to older than 76 years) exhibiting either acute (48%) or chronic (52%) pain pathologies, including headache (8%), cervical pain (20%), joint pain (32%), muscle aches (16%), lower back pain (12%) and other pain (12%), was studied for two months and compared to different NSAIDs. The volunteers received 2 capsules/day containing 100 mg of PipeNig® (corresponding to 30 mg of bioactive BCP) for 10 days or 2 capsules/day until pain relief. A score was recorded for all volunteers based on a questionnaire reporting their direct experience with PipeNig®. In general, volunteers reported a 60% reduction of pain between the 3rd and the 4th day after administration (Fig. 2A) and the perceived effect was similar or slightly lower than the NSAID used for 38% and 31% of the volunteers, respectively (Fig. 2B). Finally, when volunteers were asked whether they would use regularly PipeNig®, more than 70% responded affirmatively (Fig. 2C).

Figure 3

Figure 3 Result of the pilot study performed by the Farmacia Centrale (Cambiano, Italy) on a balanced sample of 31 volunteers exhibiting either acute or chronic pain pathologies after administration of PipeNig® as compared to different NSAIDs. A, timing of pain reduction after administration of PipeNig®. B, perception of the effect of PipeNig® as compared to the NSAID usually taken to reduce pain. C, response of volunteers to the question whether they would use again PipeNig® as a pain-killer. Values are expressed as percentage of responses.

The careful authentication the bioactive BCP by GC-MS and the quantification and standardization by GC-FID are necessary to prepare effective doses for analgesic activity. Our pilot study indicates PipeNig®-PWD and PipeNig®-FL as attractive candidates for the development of novel natural painkiller preparations for the reduction of the most distressing pain pathologies.

The recommended dosage of PipeNig®-PWD is 100 mg/dose. This dosage has been demonstrated to be effective when administered once a day for at least 10 days.

PipeNig®-FL is an alcohol-free liquid source with the highest content of (800 g/kg standardized BCP) available on the market and is particularly suitable for all liquid applications, including softgels. PipeNig®-FL suggested dosage is 30-50 mg/dose.

For further reading on PipeNig® refer to the publication:

Maffei, M. (2018)
PipeNig®, a black pepper (Piper nigrum) extract with a standardized content of (E)-β-caryophyllene. A natural source of an endo-cannabinoid agonist of the CB2 receptor.
Nutrafoods (2018) 17:N15-N17 – (Download pdf).

PIPENIG®-FL REDUCES LIPID ACCUMULATION IN 3T3-L1 PREADIPOCYTES

BCP has been demonstrated to directly activate peroxisome proliferator-activated receptor-α (PPARα)2, involved in liver lipid metabolism, and to trigger the activation of PPAR3, involved in adipogenesis (Machado et al. 2018, Phytother. Res. 32: 2376-88).

Giving the growing scientific interest in BCP, we investigated the metabolic effects of PipeNig® (Endophyllene®), containing 80% β-caryophyllene. In particular, we focused on its potential antiobesogenic activity in in vitro cell models using 3T3-L1 preadipocytes.

PipeNig®-FL effects on 3T3-L1 adipocyte cell viability

The long-term viability of 3T3-L1 cells treated with a wide range of PipeNig®-FL concentrations (100 nM, 1 µM, 10 µM, 100 µM, 1 mM, 10 mM) was determined by the CellTiter-Glo® viability assay, a method based on measurement of ATP content, whose amount is directly proportional to the number of metabolically active cells present in culture. As shown in Figure 4, cell viability was affected only at very high PipeNig®-FL concentrations (1 mM and 10 mM), with only a minor, not statistically significant decrease at 100 μM.

figure-4-viability

Figure 4. PipeNig®-FL affects 3T3-L1 cell viability only at high (millimolar) concentrations. 3T3-L1 cells were induced to differentiate into adipocytes for 9 days and treated with increasing concentrations of PipeNig®-FL for the entire differentiation period. The bar graph summarizes cell viability based on ATP content. Data in percentage referred to control condition are represented as the mean ± standard error of the mean (SEM) of three independent experiments. *** p < 0.001 vs. control.

PipeNig®-FL reduces intracellular lipid accumulation in 3T3-L1 cells without altering the cell number

The potential antiadipogenic activity of PipeNig®-FL was assayed on the murine 3T3-L1 preadipocyte cell line, a commonly used cell model for adipose cell biology research [Kim et al. 2018, Nutrients 10]. Since antiadipogenic effects can be exerted by reducing both intracellular lipid accumulation and/or the number of adipocytes (either by decreasing cell proliferation or inducing cell death), we simultaneously assayed triglyceride accumulation (AdipoRedTM assay) and cell number (NucBlueTM staining, measuring DNA content). PipeNig®-FL reduces intracellular lipid accumulation in 3T3-L1 cells without altering the cell number. Figure 5 shows representative images of AdipoRed (red) and NucBlue (blue) staining of undifferentiated preadipocytes (UNDIFF), differentiated control adipocytes (CTRL) and 10 µM PipeNig®-FL-treated 3T3-L1 adipocytes after 9 days of differentiation.

figure-5-cells

Figure 5. Confluent preadipocytes, cultured in 96-well plates, were induced to start adipogenic differentiation and were treated throughout the differentiation period (9 days) with a vehicle only (0.1% DMSO; differentiated control) or with 1 nM, 10 nM, 1 µM, 10 µM PipeNig®-FL. Higher PipeNig®-FL doses were not used, based on the cell viability data reported above. After 9 days from the beginning of adipocyte differentiation, AdipoRedTM/NucBlueTM stainings were performed on 3T3-L1 adipocytes.

Trygliceride accumulation and DNA content were calculated as percentage change from differentiated DMSO-treated controls (Figure 6). As shown in Figure 6a, triglyceride accumulation per well was reduced in PipeNig®-FL treated 3T3-L1 cells compared to differentiated control cells; in particular, statistically significant reductions were obtained after treatment with PipeNig®-FL 10 nM, 1 µM and 10 µM. DNA content was not significantly different in PipeNig®-FL treated cells and control cells, thus indicating that the decrease in lipid accumulation exerted by PipeNig®-FL is not due to a decrease in cell proliferation or to cytotoxic effects (Figure 6b). On the other hand, a significant reduction in intracellular lipid content per cell was found at all PipeNig®-FL concentrations (Figure 6c).

figure-6-lipids

Figure 6. (a) Bar graph summarizing AdipoRed staining experiments to assess lipid accumulation on undifferentiated cells, differentiated control and 3T3-L1 adipocytes treated with various concentrations of PipeNig®-FL for 9 days, showing triglyceride accumulation per well. (b) Bar graph summarizing NucBlue staining experiments to assess variations in the number of cells, showing DNA content per well. (c) Bar graph showing triglyceride accumulation per cell, calculated as the ratio of AdipoRed and NucBlue staining. Data in percentage referred to differentiated control condition are represented as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

The development of obesity is characterized by an increase in the number of fat cells (hyperplasy) and their lipid content (hypertrophy), as a result of cell proliferation and differentiation. In our experiments PipeNig®-FL led to a significant decrease in the lipid content per cell, without affecting cell proliferation, thus suggesting a role in reducing adipocyte-hypertrophic response typically present in the energy overload conditions that characterizes metabolic syndrome [Hafidi et al. 2019, Int. J. Mol. Sci. 20, 3657]. Our results of reduced adipogenesis induced by PipeNig®-FL in 3T3-L1 pre-adipocytes support the effectiveness of the extract due to its high and standardized BCP content.

PIPENIG®-FL IMPROVES GLUCOSE UPTAKE AND ACTS AS A INSULINOMIMETIC IN C2C12 SKELETAL MUSCLE CELLS

The pharmacological treatment of metabolic syndrome commonly involves anti-obesity drugs, thiazolidinediones (TZDs), metformin, statins, fibrates and several other drugs [Ammazzalorso et al. 2019, Eur. J. Med. Chem. 173, 261–273], but its management chiefly lies in the adoption of a healthy lifestyle [Aguilar-Salinas and Viveros-Ruiz 2019, F1000Research 8]. BCP has been highlighted as a hypocholesterolemic and insulinotropic agent in high-fat diet-fed [Youssef et al. 2019, Chem.-Biol. Interact. 297, 16–24], or in streptozotocin-induced, diabetic rats [Basha and Sankaranarayanan 2016, Chem. Biol. Interact. 245, 50–58], where it showed non-clinical toxicity and an absence of adverse effects [da Silva Oliveira et al. 2018, Regul. Toxicol. Pharmacol. 92, 338–346]. BCP may represent a promising treatment for several metabolic disorders.

PipeNig®-FL does not affect cell viability on C2C12 muscle cell

C2C12 differentiated myotube cells were treated with increasing concentration of PipeNig®-FL (100 nM, 50 µM, 200 µM, 1 mM, 10 mM) for 1 h. Acute exposure (1 h) to PipeNig®-FL even at the highest doses did not affect significantly (p < 0.05) cell viability (Figure 7).

figure-7-viability-pipenig

Figure 7. PipeNig®-FL does not have any effects on cell viability in C2C12 muscle cell. C2C12 cells were treated with increasing concentration of PipeNig®-FL for 1h. Data in percentage referred to control condition and are represented as the mean ± SEM (n = 3)

PipeNig®-FL improves glucose uptake in C2C12 myotubes

To verify the potential role of PipeNig®-FL on glucose uptake in skeletal muscle cells, we performed confocal laser scanning microscopy analyses by using a fluorescent D-glucose analog tracer used for monitoring glucose uptake into living cells, 2-NBDG [D-Glucose, 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-]. Cells were incubated simultaneously with either 100 µM 2-NBDG and different concentrations of PipeNig®-FL (1–10–100 nM) without insulin while insulin alone (25 nM) was used as positive control, for 30 min in the dark. A significant increase of glucose uptake was observed in either PipeNig®-FL or insulin treated cells with respect to control cells, whereas no differences were observed between insulin and PipeNig®-FL treatments or among PipeNig®-FL concentrations (Figure 8). These results indicated that PipeNig®-FL acts as a insulinomimetic extract.

figure-8-insulin

Figure 8. PipeNig®-FL stimulates glucose uptake. (A) Representative confocal images of C2C12 skeletal muscle cells (myotubes) incubated with the fluorescent glucose analog 2-NBDG for 30 min. Images are presented in pseudocolor (LUT = fire) to better show the fluorescence intensity variations. Insulin (25 nm) was used as a positive control. (B) Bar graph summarizing the experiments of fluorescent glucose uptake. Data in percentage referred to control condition are represented as the mean ± SEM (n = 4). * p < 0.05; ** p < 0.01 vs. control.

PipeNig®-FL induces GLUT4 translocation in the plasma membrane of skeletal muscle C2C12 cells and acts as insulinomimetic

To confirm the involvement of PipeNig®-FL on glucose metabolism, we carried out immunofluorescence experiments using glucose transporter type 4 (GLUT4) antibody, followed by a detailed image analysis of peripheral vs. internal fluorescence staining. GLUT4, also known as facilitated glucose transporter member 4, it is a human protein and is the insulin-regulated glucose transporter found primarily in adipose tissues and striated muscle (skeletal and cardiac) [Morgan et al. 2011, Recent. Pat. Endocr. Metab. Immune. Drug Discov. 5, 25–32].

Cells were treated with either insulin (25 nM) or different concentrations of PipeNig®-FL (1–10–100 nM) without insulin, for 30 min. An evident translocation of the glucose transporter from the cytosol to the plasma membrane was observed in both insulin and PipeNig®-FL treated cells with respect to control cells, whereas there were no significant differences in staining among treatments (Figure 9). These data confirm the potential insulinomimetic effect of PipeNig®-FL.

figure-9-glucose

Figure 9. PipeNig®-FL induces GLUT4 translocation to the plasma membrane. (A) Confocal images of GLUT4 immunofluorescence staining. After PipeNig®-FL stimulation (1–10–100 nM) the fluorescent signal is clearly localized to the peripheral plasmalemma, thus indicating the GLUT4 translocation. Images are presented in pseudocolor (LUT = fire) to better show the fluorescence intensity variations. Insulin (25 nM) was used as a positive control. (B) Bar graph representing the ratio peripheral vs. internal GLUT4 fluorescence intensity. Data are represented as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0,001 vs. control.

These results indicate that PipeNig®-FL is as efficient as insulin in stimulating in vitro cellular glucose uptake. Our results are in agreement with previous studies reporting antidiabetic properties of BCP through an enhanced insulin release [Suijun et al. 2014, Biochem. Biophys. Res. Commun. 444, 451–454]. Moreover, PipeNig®-FL-treated myotubes show a significant plasma membrane GLUT4 translocation with respect to control. Thus the attractive novelty of these results is that PipeNig®-FL, a BCP-high content and standardized natural extract, promotes in vitro glucose uptake in skeletal myotubes, likely through an improvement of GLUT4 trafficking toward the plasma membrane.

Future experiments are under way to gain further information on the molecular mechanisms initiated by PipeNig®-FL and to confirm its properties as anti-lipidogenic and glucose uptake inducer in animal models.

For further reading on PipeNig® antiobesogenic and insulinomimetic activity refer to the publication:

 

Geddo F., Scandiffio R., Antoniotti S., Cottone E., Querio G., Maffei M., Bovolin P. e Gallo M.P. (2019)
PipeNig®-FL, a fluid extract of black pepper (Piper nigrum L.) with a high standardized content of trans-β-Caryophyllene, reduces lipid accumulation in 3T3-L1 preadipocytes and improves glucose uptake in C2C12 myotubes
Nutrients 2019, 11 – (read pdf)

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