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Article

Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer

by
Ingryd Nayara de Farias Ramos
1,*,
Monique Feitosa da Silva
1,
Jefferson Marcio Sanches Lopes
2,
Jordy Neves Cruz
3,
Fabrine Silva Alves
4,
José de Arimatéia Rodrigues do Rego
5,
Marcondes Lima da Costa
6,
Paulo Pimentel de Assumpção
1,
Davi do Socorro Barros Brasil
3,4,5 and
André Salim Khayat
1,7
1
Oncology Research Center, Federal University of Pará, Belém 66075-110, PA, Brazil
2
Department of Physics, Federal University of Roraima, Boa Vista 69310-000, RR, Brazil
3
Institute of Technology, Federal University of Pará, Belém 66075-110, PA, Brazil
4
Graduate Program in Pharmaceutical Innovation, Federal University of Pará, Belém 66075-110, PA, Brazil
5
Graduate Program in Science and Environment, Federal University of Pará, Belém 66075-110, PA, Brazil
6
Institute of Geosciences, Federal University of Pará, Belém 66075-110, PA, Brazil
7
Institute of Biological Science, Federal University of Pará, Belém 66075-110, PA, Brazil
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(14), 5587; https://doi.org/10.3390/molecules28145587
Submission received: 10 June 2023 / Revised: 30 June 2023 / Accepted: 5 July 2023 / Published: 22 July 2023
(This article belongs to the Special Issue Natural Products in Anticancer Activity)
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Versions Notes

Abstract

:
Gastric cancer is one of the most frequent types of neoplasms worldwide, usually presenting as aggressive and difficult-to-manage tumors. The search for new structures with anticancer potential encompasses a vast research field in which natural products arise as promising alternatives. In this scenario, piperine, an alkaloid of the Piper species, has received attention due to its biological activity, including anticancer attributes. The present work proposes three heating-independent, reliable, low-cost, and selective methods for obtaining piperine from Piper nigrum L. (Black pepper). Electronic (SEM) and optical microscopies, X-ray diffraction, nuclear magnetic resonance spectroscopies (13C and 1H NMR), and optical spectroscopies (UV–Vis, photoluminescence, and FTIR) confirm the obtention of piperine crystals. The MTT assay reveals that the piperine samples exhibit good cytotoxic activity against primary and metastasis models of gastric cancer cell lines from the Brazilian Amazon. The samples showed selective cytotoxicity on the evaluated models, revealing higher effectiveness in cells bearing a higher degree of aggressiveness. Moreover, the investigated piperine crystals demonstrated the ability to act as a good cytotoxicity enhancer when combined with traditional chemotherapeutics (5-FU and GEM), allowing the drugs to achieve the same cytotoxic effect in cells employing lower concentrations. These results establish piperine as a promising molecule for therapy investigations in aggressive gastric cancer, both in its isolated form or as a bioenhancer.

1. Introduction

Gastric cancer occupies the fifth position among the most common types of cancer worldwide [1,2]. Generally diagnosed late at advanced stages of the disease, this type of tumor inherits high heterogeneity, which is a determinant feature for its phenotypic aggressiveness [3] and high mortality rates. The limitations faced in current therapy are additional concerns since they can lead to severe side effects and resistance to multiple drugs, implying high recurrence rates and relative therapeutic failure [4,5]. This set of factors reveals that gastric cancer is still a clinically challenging disease, with low effective treatment options, especially concerning aggressive tumors [6]. Therefore, searching for more effective treatments based on new molecules is necessary for improving therapy.
Natural products are promising sources of pharmacologically active molecules capable of interacting with cell membrane receptors, nucleic acids, and other mechanistic pathways [7,8,9]. Their potential biological activity has been intensively investigated in preclinical and clinical studies [10], revealing promising effects related to a wide range of plant-derived compounds and secondary metabolites such as terpenes, phenolic compounds, and alkaloids [11]. Indeed, natural molecules such as taxol, artemisinin, and vinblastine [12] are currently employed in a variety of drugs for the clinical treatment of different diseases, being plant-derived species of particular importance [10].
Among the vast number of natural structures, piperine (Figure 1), a remarkable active alkaloid generally found in species of the Piper family, such as Piper longum (long pepper) and Piper nigrum L. (black pepper), and responsible for their pungencies, has been studied recently [13,14]. The relative simplicity of its obtention makes piperine commercially attractive. In this regard, controlling physicochemical parameters allows the establishment of different extraction approaches, such as the soxhlet method, which involves sample heating and requires a low amount of organic solvents [15], being a relatively inexpensive way for obtaining piperine. However, compared to other approaches, the soxhlet method demands a long extraction time [16]. Methods based on external agents such as ultrasound [17], microwaves [18,19], maceration [20,21], and others [22] are also reported in the literature. In all cases, piperine isolation relies on a recrystallization step, resulting in yellow-colored needle-shaped microscopic crystals obtained with good yields and high purity [16,23].
Piperine exhibits several biochemical and pharmaceutical properties, being capable of interacting with chemically and functionally diverse molecular targets [24], as confirmed by in vitro and in vivo studies. The attention attributed to the study of this molecule is mainly due to its biological properties, such as anti-inflammatory [25], antimicrobial [26,27], antidepressant [28,29], antifungal [30,31], and anticancer properties [32,33]. In addition, piperine is regarded as a target compound, as it is the principal molecule in black pepper extract and belongs to the class of alkaloids, which have already proven anticancer activity through chemical interactions and activation of different pathways in tumor cells [34]. Specifically, regarding the anticancer activity of piperine, recent observations show that its mechanism of action is multiple and involves the activation of cellular and molecular signaling pathways, with programmed cell death, decreased migration and invasion, and reduced cell proliferation [35]. Notably, in addition to acting in isolation, piperine can increase the bioavailability of other compounds [36,37], favoring its use as a food supplement [38] and in combination with drugs from different classes (including current chemotherapy agents) [39,40].
This set of factors places piperine at the heart of the search for new therapeutic approaches for gastric cancer. In the present work, we obtained piperine via three heating-independent methods and characterized the samples employing several techniques. Moreover, the molecule’s in vitro cytotoxic activity in its isolated form and combined with the chemotherapeutics 5-Fluorouracil (5-FU) and Gemcitabine (GEM) are evaluated. The cytotoxicity experiments were counted with five Brazilian Amazon native gastric cancer lineages (AGP01, AGP01 PIWIL1 KO, ACP02, and ACP03), with the models bearing a variety of molecular profiles, including metastatic biomarkers and genetic modifications [41]. We present novel discussions on the in vitro cytotoxicity of piperine against different gastric cancer lineages, demonstrating the relationship between the molecule’s cytotoxic activity and the phenotypes of the evaluated models. The results show that piperine has pronounced activity against cells with a higher level of aggressiveness. In addition, we also showed that besides acting in isolation in cancer models, piperine enhances the activity of commercial chemotherapy drugs.

2. Results and Discussion

2.1. Isolation and Characterization of Piperine

2.1.1. Microscopy and X-ray Diffraction Analysis

The recrystallization of piperine from Piper nigrum L. extracts employing the three heating-independent approaches proposed in this work revealed reliable and low-cost methodologies. The obtained solid-state yellow-colored samples (P1, P2, and P3), shown in Figure 2, exhibited good structural and color stability at room temperature for at least 120 days of storage.
Optical microscopy analysis of these samples (Figure 2D–F) revealed that they are composed of needle-shaped crystals, which along with their characteristic yellow color are compatible with the reported piperine crystal [26,42,43]. Comparing the micrograph of P3 with the results from P1 and P2, we observe that although aggregated, this sample is still composed of needle-shaped crystals. This result confirms that adopting only pure water as a recrystallization mediator is still effective for obtaining the piperine crystals, even without employing KOH in the process.
As discussed later, further characterization with other techniques (e.g., XRD, NMR, and FTIR) reveals no significant differences between the P1, P2, and P3 samples. This trend led us to employ P2 as a representative for the other samples in the scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis. In agreement with the literature [26,44,45], in Figure 3A, it is demonstrated that the crystals in P2 follow the monoclinic system, with longitudinal lengths varying between ~100 μm and ~800 μm. The EDS analysis (Figure 3B) reveals that the sample is mainly composed of carbon (70.0% of relative weight) and oxygen (26.9% of relative weight) present in abundance in piperine which is supportive of its level of purity.
The XRD analysis reveals several atomic planes in all samples, with peaks identified between 10° and 50° (Figure 4). The observed diffractograms agree with the literature supporting the assignment of a monoclinic regime presented in piperine crystals [26,44,45]. As shown in Figure 4C, the XRD diffractogram of sample P3 exhibits an increased baseline, which is suggestive of sample amorphousness and can correlate with the crystal aggregation observed by optical microscopy.

2.1.2. Spectroscopy Analysis

Employing NMR spectroscopies (13C and 1H), we could track signatures from piperine’s aromatic, amide, and aliphatic groups in all samples (P1, P2, and P3). Firstly, considering the 13C NMR spectra, depicted in Figure 5A–C, we verify that the π carbons of the aromatic portion of piperine can be assigned [42,46,47] to the transitions located at ca. 120 ppm, 123 ppm, and 144 ppm, while the O-C-O and C=C-O bonds correlate with the transition at ~125 ppm and the transitions at ~28 ppm, ~45 ppm, and ~48 ppm, respectively. The amide portion of piperine [42,46,48] is associated with the transitions at ~100 ppm (N-C=O), ~109 ppm, and ~150 ppm. Finally, the transitions located at ~132 ppm, ~139 ppm, and 166 ppm correlate with the aliphatic chain [42,47,49]. The transition at ~78 ppm is assigned to the reference solvent (CDCl3).
The same set of functional groups is observed in the 1H NMR spectra, as shown in Figure 5D–F. The aromatic moiety is related to the transitions located at ca. 6.6 ppm, 7.3 ppm, 1.68 ppm (C=C-O), 1.73 ppm (C=C-O), and 3.6 ppm (C=C-O) [42,46,47]. The transition at ~5.9 ppm is characteristic of the N-C=O bond, which addresses the presence of the piperine’s amide moiety. The signatures at ~6.8 ppm and ~7.1 ppm evidence the aliphatic chain. The signal associated with the reference solvent (CDCl3) is located at ~7.5 ppm. According to all the presented results, there are no significant differences between the NMR spectra of the P1, P2, and P3 samples. Furthermore, no additional signals, other than the transitions specific from piperine and the adopted solvent, were observed, suggesting that if impurities are present in the crystals, they display relatively small concentrations regarding the concentration of piperine.
The vibrational spectra of P1, P2, and P3 share significant similarities (Figure 6), with all samples bearing vibrational modes located at 1033 cm−1 (symmetric =C-O-C stretching), 1132 cm−1 (asymmetric =C-O-C stretching), and 1580 cm−1 (aromatic stretching of the benzene ring) [18,47,50] which identify the aromatic portion of the piperine structure. The amide portion is associated with the vibrational mode at 1631 cm−1 (C=O-N bond) [46,51]. Finally, the high-energy vibrational mode centered at 2940 cm−1 [42,46,47] assigns the C-H stretching in the aliphatic chain.
Since we measured the samples in their pristine form (solid-state), FTIR spectroscopy also allows tracking further information on the presence of possible remnant undesired substances. Of particular concern, the recrystallization of piperine could trap remnant reagents such as water and KOH inside or outside the solid samples. As reported in the literature, the water molecule exhibits active vibrational modes at ~3300 cm−1, correlated with the symmetric and asymmetric stretching of its O-H bond [52]. Regarding KOH, Snyder and co-workers [53] demonstrated that at room temperature (~23 °C), a broadband vibrational mode at ~3600 cm−1 arises from its O-H stretching. After conducting a careful evaluation in the high energy region of our spectra, we verified the existence of low-intensity signals at ~3307 cm−1 and 3404 cm−1 (Figure 6b) assigned to the vibrational modes of water [52]. Notably, the low intensity of these peaks regarding the piperine’s vibrational modes suggest reduced amounts of moisture in all samples. No evidence of KOH signatures is verified, endorsing the absence of remnant reagents in the samples.
From UV–Vis absorption spectroscopy, we verify that P1, P2, and P3 display characteristic absorption bands centered at 299 nm, 311 nm, and 345 nm, respectively (Figure 7A). These features are consistent with previously reported data [54,55,56] and can be associated with electronic and vibronic transitions in piperine. The steady-state photoluminescence (PL) spectra of the samples display a characteristic emission band centered at 440 nm, which reportedly arises from piperine [57] (Figure 7B). Notably, no other emission bands are observed in Figure 7B, suggesting that piperine is the only emissive compound in P1, P2, and P3 samples. Comparing the most intense absorption and PL bands, we verify Stokes shifts of ~6260 cm−1 in all samples, which is in agreement with the literature [57].

2.2. Piperine Cytotoxicity in Gastric Cancer Models

2.2.1. Isolated Piperine

The cytotoxicity of the P1, P2, and P3 samples was evaluated for the AGP01 lineage, which originates from peritoneal metastasis and has genetic alterations capable of providing more resistance to the action of substances or chemotherapy [58,59,60]. As observed by the MTT assay, all piperine samples are cytotoxic against the tested model, with IC50 values of 16.81 µg/mL (P1), 12.06 µg/mL (P2), and 16.69 µg/mL (P3), as shown in Figure 8. Furthermore, P1, P2, and P3 caused a decrease in cell viability as a function of the increase in concentration, demonstrating a concentration dependency. The results also show that P2 displays higher cytotoxic activity, with a lower IC50 value and with a statistically significant reduction in cell viability, starting from a concentration of 12.5 µg/mL (Figure 8B). Other studies have also demonstrated that piperine causes a concentration-dependent reduction in cell viability of rectal [61], cervical [62], and prostate cancer cells [63]. However, the IC50 of piperine in these studies lies around 30 µg/mL, showing that piperine is even more active in metastatic gastric cancer.
From the initial screening, the P2 sample (which showed the best activity among all piperine samples) was evaluated for cytotoxicity in three other gastric cancer lineages: a diffuse model (ACP02), an intestinal model (ACP03), and a metastasis model with an inactivated PIWIL1 gene (AGP01 PIWIL1 KO). Additionally, aiming to investigate the selectivity of the compound, a non-tumor cell line (VERO) was also evaluated. The results show that P2 is cytotoxic in all tested models. However, there are very significant differences in the obtained IC50 values. These values were 44.32 µg/mL, 26.28 µg/mL, 47.10 µg/mL, and 43.44 µg/mL for AGP01 PIWIL1 KO, ACP02, ACP03, and VERO lineages, respectively (Figure 9). The level of cell viability was also heterogeneous between the cell models, with most of them showing significant differences regarding the negative control only in the two highest tested concentrations (50 and 100 µg/mL) (Figure 9A,C,D). Especially for ACP02, this difference starts from 25 µg/mL (Figure 9B).
According to the results, AGP01 (Figure 8B) is the most sensitive cell to treatment with P2, presenting a lower IC50 when compared to the other tested models. Furthermore, this lineage is the only selective, regarding the non-tumor model (VERO), with a selectivity index (SI) [64,65] of 3.6, demonstrating that P2 has specific mechanisms in the tumor cells. The selective action of piperine has been reported for other cancer models, with higher cytotoxicity against tumor cells compared to normal ones [33,66]. These results show that piperine is safe for therapeutic use, being even advantageous over other non-selective tested molecules and substances of clinical use [67,68].
Multiple factors can explain this behavior, such as the intrinsic pathways of different lineages under the action of piperine. Data from the literature have already demonstrated that piperine displays distinct mechanistic actions, mainly attributed to its ability to interact with different molecular targets, including kinases [32], transcription factors [69], cell cycle proteins [70], receptors, and molecules of signaling [71], thus supporting its potential as an anticancer agent. Among the studied effects of piperine, both in vitro and in vivo, are included the induction of apoptosis [72,73], the inhibition of cell proliferation [63,74] with cell cycle arrest [70,73], and the modulation of the expression of genes and proteins involved in the processes of cell migration and invasion [75,76]. All these effects influence the outcome of carcinogenesis of various types of tumors, such as breast [77,78], cervical [62,79], and colorectal [80,81].
Considering that the IC50 of P2 on the AGP01 lineage (12.06 µg/mL) was almost four times lower than on the AGP01 PIWIL1 KO lineage (44.32 µg/mL), we can attribute such better cytotoxicity to the inactivation of the PIWIL1 gene, since that is the only difference between the lineages. According to the literature, the inactivation of the PIWIL1 gene is associated with a decrease in the cell’s ability to migrate and invade, promoting a change in the gene expression related to these processes [82,83]. Therefore, considering the higher cytotoxicity of P2 in AGP01, which has all these genes activated, and the data reported on the action of piperine in other types of tumors [33,84,85], it is possible to suggest that the effects of piperine in gastric cancer may be closely related to the presence of specific pathways of cell migration and invasion. Moreover, piperine seems more effective for treating tumor cells with high levels of aggressiveness.
Regarding the action of P2 in primary gastric cancer models (ACP02 and ACP03), the cytotoxicity was higher in the diffuse type model (ACP02; IC50: 26 µg/mL) concerning the intestinal type (ACP03; IC50: 47 µg/mL). Diffuse gastric cancer (CGD) exhibits a more aggressive characteristic, with undifferentiated cells, mutations, and changes in the expression of genes involved in the epithelial-mesenchymal transition (EMT) [86,87,88]. In this scenario, we can infer that the more pronounced cytotoxicity of P2 on the ACP02 lineage once again shows that piperine has a better action on cancer cells with a more aggressive phenotype and that possibly its mechanism of action is involved with the migration and invasion pathways, supporting its potential use. Corroborating our results, Gunasekaran and co-workers showed that piperine has a remarkable in vitro action on hepatocellular carcinoma, an aggressive and difficult-to-treat tumor [89]. In breast cancer with a triple-negative phenotype, a similar result has been reported, with piperine showing antiproliferative activity [90]. This tumor is considered one of the most aggressive breast cancers due to its rapid growth and increased probability of generating metastasis [91], which endorses our hypothesis that piperine is more active in more aggressive cells capable of expressing migration and invasion pathways.
Following the results, new opportunities for evaluating therapeutic interventions in gastric neoplasms with aggressive phenotype arise, which is appropriate since they are diagnosed later, with a worse prognosis and with high mortality rates [92,93]. However, further investigations regarding the intracellular and molecular mechanisms by which piperine acts in gastric cancer are necessary for complete elucidation.

2.2.2. Piperine in Association with 5-Fluorouracil and Gemcitabine

The fact that piperine has shown increased activity in cells with a more aggressive phenotype, i.e., characterized by high levels of mutations, genomic instability, and the improved ability to grow and spread [94,95], is associated with its already reported capacity to enhance the activity of other drugs opens promising opportunities. Therefore, we evaluated the cytotoxic activity against the metastatic lineage AGP01 by combining piperine with two chemotherapeutics of clinical use in gastric cancer, 5-Fluorouracil (5-FU) and gemcitabine (GEM).
Regarding 5-FU, the results show that this chemotherapeutic displays concentration-dependent cytotoxicity in the tested model, leading to a reduction in its cell viability, with a significant difference in comparison to the negative control, starting at the lowest concentration tested (0.313 µg/mL). As present in Figure 10A, the viability percentage at this concentration is approximately 90%, reaching 50% only at the concentration of 2.5 µg/mL. This result shows that at lower 5-FU concentrations there is only a small variation in cell viability, with great variations requiring the highest tested concentrations.
Combining 5-FU prepared in the same concentration range (0–10 µg/mL) with a single concentration of piperine (P2), set to its IC50 value (~12 µg/mL), we observed a significant improvement in the concentration-dependent cytotoxic effect of 5-FU. As depicted in Figure 10B, the combination improves the activity of 5-FU, allowing the lowest concentration of the chemotherapeutic (0.313 µg/mL) to decrease cell viability to 50% in the presence of piperine. Since in the absence of piperine, the viability only reaches 50% for 2.5 µg/mL, this trend demonstrates that the combination effectively enhances the action of 5-FU, allowing the same cytotoxic effect in cells even employing concentrations approximately eight times lower.
The cytotoxicity evaluation of GEM followed a similar behavior. As shown in Figure 10C, GEM is cytotoxic to the AGP01 lineage over the entire concentration range tested (0–10 µg/mL). For the lowest concentration tested (0.313 µg/mL), cell viability reaches 70%. As the concentration increases, a saturation pattern is achieved (Figure 10C). However, the combination of piperine with GEM significantly enhances the cytotoxic effect. As depicted in Figure 10D, the viability of the lower concentration of GEM (0.313 µg/mL) decreases to approximately 30% in the presence of piperine. Although viability still saturates with the increase of concentration, we verify that in the combination test, the limit value is lower (~12%) concerning the viability found in the absence of piperine (~50%).
The combination with P2 increases the cytotoxic activity of GEM so significantly that the effect on gastric cancer cells at the lowest concentration of the combined curve (0.313 µg/mL) is even greater than that of the highest concentration (10 µg/mL) in the absence of piperine, where the cell viability values are 32.75% and 47.3%, respectively. This evidence shows that piperine can improve the action of GEM in gastric cancer cells considerably and that the combined treatment could decrease the employed concentration of the chemotherapeutic, causing better effects than in its isolated form.
One of the main disadvantages of current chemotherapy for gastric cancer is the increased toxicity of drugs, especially at high concentrations [96,97]. Therefore, evaluating new substances able to increase their effects at lower doses would eventually reduce this toxicity [98,99,100]. The combination of drugs in cancer therapy is a valuable treatment modality, as it increases the effectiveness of drugs, seeking to act on the main pathways in a synergistic or additive way [101]. This approach can decrease resistance to chemotherapy and provide anticancer benefits such as reduced cell proliferation and metastasis [102]. Thus, the results observed for the combination between piperine, and the chemotherapy drugs 5-FU and GEM corroborate the data already reported in the literature, which show significant effects of piperine in increasing the bioavailability of other compounds, such as antitubercular compounds [103], anti-inflammatory compounds [104], antibiotics, and even chemotherapeutics [105]. These reports demonstrate that different systems combined with piperine can have a higher impact on the bioavailability of co-administered drugs by diverse mechanisms, including inhibiting efflux transport, inhibiting intestinal and hepatic metabolism, and modulation of activity and expression of metabolic response enzymes [106].
The action of piperine combined with chemotherapeutic agents for clinical use in other types of cancer has also been reported. In breast cancer cells, piperine was combined with cisplatin and led the cells to apoptosis more effectively than the drug administered alone, reducing the toxic dose used in chemotherapy [40]. The combination of piperine and docetaxel brought an improved antiproliferative response against taxane-resistant prostate cancer in vitro and in vivo models [107,108]. Furthermore, in an in vitro model of cervical cancer (HeLa linage), piperine combination with paclitaxel demonstrated a synergistic effect, sensitizing tumor cells toward the drug’s action [109]. Therefore, chemotherapeutic systems combined with piperine are effective in the cytotoxicity of tumor cells, generating increased absorption and therapeutic efficacy of the drugs and allowing lower concentrations to produce the same or even improved effects [71,110,111]. This trend endorses our findings in gastric cancer models since piperine enhanced the activity of the tested chemotherapeutic drugs, decreasing their required effective concentrations in vitro. Because piperine exhibits multiple mechanisms in gastric cancer cells, its enhancing ability may still bring other benefits in the search for more effective treatments.

3. Materials and Methods

3.1. Reagents and Chemicals

Potassium hydroxide (KOH) flakes (≥90.0%), P.A. ethyl alcohol (≥99.9%), and P.A. deuterated chloroform (CDCl3) (≥99.8%) were purchased from Êxodo Científica (Sumaré, São Paulo, Brazil). The 5-fluorouracil (≥99.9%) and Gemcitabine (≥98.0%) chemotherapeutics, the P.A. dimethyl sulfoxide (DMSO) (≥99.5%), and the MTT salt (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium) were purchased from Sigma-Aldrich/Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Penicillin (100 U/mL)/Streptomycin (10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin) were purchased from Gibco® (Grand Island, NY, USA). All reagents and chemicals were used as received with no further purification processing.

3.2. Plant Material and Obtention of Piperine Crystals

Dried seeds of black pepper (Piper nigrum L.), purchased from a local market of Abaetetuba city in the state of Pará, Brazil, were milled mechanically employing a porcelain crucible and pestle arrangement (A-100, Chiarotti, Mauá, São Paulo, Brazil). Afterward, we immersed 200.0 g of the crushed black pepper into 1.0 L of P.A. ethyl alcohol. The solution was stored at room temperature (~30 °C) for seven days until submitted to simple filtration to remove remnant milled pepper, resulting in the ethanolic extract of Piper nigrum (EEPN).
Considering that isolating natural molecules from extracts is generally a difficult endeavor in which undesired products (e.g., essential oils, isomers, and other metabolites) can be obtained concomitantly, we opted to employ three heating-independent recrystallization-based isolation methodologies to force piperine to crystallize and form solid-state samples (P1, P2, and P3). The obtention of P1 counts with the mixing, at room temperature, of a 100.0 mL EEPN aliquot with 10.0 mL of an aqueous KOH solution (4% v/v) and 40.0 mL of distilled water. The sample P2 followed a similar methodology, mixing a 100.0 mL EEPN aliquot with 10.0 mL of an ethanolic KOH solution (4% v/v) and 40.0 mL of distilled water. Finally, sample P3 originates from a green extraction method, free of KOH, in which a 100.0 mL EEPN aliquot is mixed directly with 50.0 mL of distilled water. All three mixtures were stored at room temperature for seven days forming yellow-colored solid precipitates after this period. The samples were filtered, and their solid phases (piperine crystals) were isolated for further characterization. Since there are no reports demonstrating that other components available in Piper nigrum L. extracts can produce yellow-colored needle-shaped crystals, we expect these substances to remain in the liquid phase, discarded after sample filtration. Six replicates were prepared to ensure the reproducibility of the proposed isolation methods.

3.3. Characterization Methods

3.3.1. X-ray Diffraction (XRD)

The X-ray diffractograms were obtained with the samples in the solid-state in a BRUKER (Leipzig, Germany) diffractometer model D2 PHASER, equipped with a goniometer (θ/θ), radius: 141.1 nm, a copper anode ceramic X-ray tube (Cu-Kα1), and a 1D Lynxeye detector with a 5°, 2θ aperture and 192 channels. The characteristic emission line is located at 1.540598 Å/8.047 keV, with a maximum power of 300 W (30 kV × 10 mA).

3.3.2. Optical Microscopy and Scanning Electron Microscopy (SEM) Coupled with Energy-Dispersive X-ray Spectroscopy (EDS)

Scanning electron micrographs were obtained for the solid-samples in a SEM microscope model TM 3000 Hitachi (Tokyo, Japan) coupled with a Energy-Dispersive X-ray Spectroscopy (EDS) TESCAN S8000 detector (Brno, Czech Republic), model VEGA TC. Optical microscopy analysis of the solid samples was performed employing a phase contrast optical microscope, Axio Observer 5 Zeiss (Oberkochen, Germany).

3.3.3. Nuclear Magnetic Resonance (NMR)

The 1H and 13C NMR spectra were obtained using an instrument from Bruker (Leipzig, Germany), model Advance 400, which is equipped with a 5 mm cryogenic probe with 16 acquisitions each at a temperature of 25.7 °C. For the measurements, 30.0 mg of the P1, P2, and P3 samples were dissolved in deuterated chloroform (CDCl3). This solvent was used as an internal reference for the calibration of the equipment. The spectral widths for 1H and 13C were 15 ppm and 200 ppm, respectively.

3.3.4. Infrared Spectroscopy

The infrared spectra of the solid-state P1, P2, and P3 samples were measured in a Fourier infrared spectrometer from Bruker (Leipzig, Germany), model Vertex 70v, which is equipped with a high efficiency interferometer (spectral resolution of ~1.0 cm−1) and a vacuum pump detector.

3.3.5. UV–Vis Absorption and Photoluminescence (PL) Spectroscopies

Absorption spectra were acquired in a JASCO V-670 spectrophotometer (Easton, MD, USA) whereas steady-state photoluminescence (PL) spectra were measured in a Deltaflex TCSPC Lifetime Fluorometer from Horiba (Kyoto, Japan), equipped with a pulsed excitation source (λexc = 352 nm with 8.0 MHZ of repetition rate). All measurements were conducted in quartz cuvettes (1.0 cm path length) with the P1, P2, and P3 samples dissolved in P.A. dimethyl sulfoxide (DMSO).

3.4. In Vitro Cytotoxicity Activity

3.4.1. Cell Culture

Gastric adenocarcinoma cell lines of the intestinal and diffuse Lauren’s types [112,113] were used, including AGP-01 (malignant ascites), AGP-01 PIWIL1 KO (PIWIL1 gene inactivated) [82], ACP02 (primary cancer of the diffuse type), and ACP03 (primary cancer of the intestinal type), as well as non-neoplastic African Green Monkey Kidney (VERO) cell for comparison. Cells were grown in adherent monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) and maintained at 37 °C in 5% carbon dioxide.

3.4.2. Cytotoxicity Assay

The cytotoxic activity of the P1, P2, and P3 samples was evaluated for all lineages (AGP01, AGP01 PIWIL1 KO, ACP02, and ACP03) using the MTT colorimetric assay. The 5-fluorouracil (5-FU) and gemcitabine (GEM) chemotherapeutics were evaluated for AGP01 lineage. This assay is based on the conversion of the yellow MTT salt to formazan, a purple chromogenic product [114,115,116] by metabolically viable cells. All solid-state samples (P1, P2, and P3) were dissolved in dimethyl sulfoxide for the MTT assay. Cells were seeded in 96-well plates at a density of 103 cells/well for 24 h to allow adhesion in the plate. The treatment was accomplished in a dose–response curve, with seven concentrations ranging from 1.56 μg/mL to 100 μg/mL, with further incubation at 37 °C for 72 h. The dissolved piperine samples in DMSO were added to the DMEM culture medium for cell treatment. The negative control was taken as the untreated cells, and the experiments were performed in triplicate. After treatment, 100 μL of MTT solution, 5 mg/mL stock solution, diluted 1:10 v/v in DMEM medium, was added to each well of the plate and incubated at 37 °C for 3 h. Absorbance of each plate was measured using a microplate spectrophotometer at 570 nm (SYNERGY/HT microplate reader, BioTek, Winooski, VT, USA).
Considering the individual IC50 values of P2 piperine (selected after the results analyses), of 5-FU and GEM, different concentrations of the chemotherapeutics (0.313–10 μg/mL) were combined to a constant concentration (IC50) of P2 (12 μg/mL), and the cell viability was obtained through of the MTT assay as described above.

3.4.3. Data Analysis

A sigmoidal dose–response equation (non-linear regression) was used to determine the half maximal inhibitory concentration (IC50) and their respective confidence intervals (95% CI). Cell viability was obtained from the percentages relative to the negative control, using Equation (1), where Absexp and Absctr account for the absorbance tracked at 570 nm for the experimental and control samples, respectively. To verify differences between the experimental groups, the ANOVA test (two way), followed by Bonferroni’s posttest, was performed, with significance levels p > 0.005 (**) and p > 0.0001 (***).
C e l l   v i a b i l i t y   % = A b s e x p A b s c t r ∗ 100

4. Conclusions

In summary, in the present study, three heating-independent extraction methods proved to be low-cost alternatives and reliable for the easy extraction of piperine crystals from black pepper seeds. These processes introduce new approaches to obtaining piperine. The samples originating from these processes (P1, P2, and P3) showed cytotoxic effects on gastric cancer cells, with P2 exhibiting the best cytotoxicity and selectivity. Furthermore, piperine was more active in cells with an aggressive phenotype, and possibly its mechanism of action involves cell migration and invasion pathways. This finding demonstrates for the first time the relationship between piperine cytotoxic activity and the phenotype of the gastric cancer lineages. Additionally, the results showed that besides acting in isolation on cancer models, when tested in combination, piperine provides considerable improvement in the activity of commercial chemotherapeutics such as 5-Fluorouracil and Gemcitabine, which is a very promising finding with possible outcomes in future research and clinical endeavors. This action leads to a decrease in the effective concentration of chemotherapeutic drugs on cells, which may mean more effective therapeutic perspectives and a reduction in toxicity. This set of results demonstrates the potential use of piperine in both isolated and combined forms in gastric cancer models, supporting the promising applicability of natural products in generating new therapies.

Author Contributions

Conceptualization, A.S.K. and I.N.d.F.R.; methodology, I.N.d.F.R., M.F.d.S., F.S.A., J.M.S.L., and J.N.C.; validation, J.d.A.R.d.R., M.L.d.C., D.d.S.B.B., P.P.d.A., and A.S.K.; formal analysis, I.N.d.F.R., J.M.S.L., J.d.A.R.d.R., J.N.C., and A.S.K.; investigation, I.N.d.F.R., J.M.S.L., M.F.d.S., F.S.A., J.N.C., M.L.d.C., J.d.A.R.d.R., D.d.S.B.B., P.P.d.A., and A.S.K.; resources, J.d.A.R.d.R., M.L.d.C., D.d.S.B.B., P.P.d.A., and A.S.K.; data curation, I.N.d.F.R. and J.M.S.L.; writing—original draft preparation, I.N.d.F.R., J.M.S.L.; F.S.A., J.d.A.R.d.R., and J.N.C.; writing—review and editing, I.N.d.F.R., J.M.S.L., and A.S.K.; supervision, A.S.K., P.P.d.A., and D.d.S.B.B.; project administration, A.S.K.; funding acquisition, P.P.d.A., A.S.K., and D.d.S.B.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ). The APC was funded by Universidade Federal do Pará/Propesp/PROGRAMA DE APOIO À PUBLICAÇÃO QUALIFICADA—PAPQ- EDITAL 01/2023.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are grateful to Newton Martins Barbosa Neto and Waldomiro Gomes Paschoal Junior from the Graduate Program in Physics of the Federal University of Pará, for granting access to their experimental facilities.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Sample Availability

Not applicable.

References

  1. Rawla, P.; Barsouk, A. Epidemiology of Gastric Cancer: Global Trends, Risk Factors and Prevention. Przegla̜d Gastroenterol. 2019, 14, 26. [Google Scholar] [CrossRef] [PubMed]
  2. Morgan, E.; Arnold, M.; Camargo, M.C.; Gini, A.; Kunzmann, A.T.; Matsuda, T.; Meheus, F.; Verhoeven, R.H.A.; Vignat, J.; Laversanne, M.; et al. The Current and Future Incidence and Mortality of Gastric Cancer in 185 Countries, 2020–2040: A Population-Based Modelling Study. eClinicalMedicine 2022, 47, 101404. [Google Scholar] [CrossRef] [PubMed]
  3. Duarte, H.O.; Gomes, J.; Machado, J.C.; Reis, C.A. Gastric Cancer: Basic Aspects. Helicobacter 2018, 23, e12523. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Liang, X.; Zhu, J.; Li, Y.; Xu, Y.; Chen, K.; Lv, L.; Mao, W. Treatment Strategies for Metastatic Gastric Cancer: Chemotherapy, Palliative Surgery or Radiotherapy? Futur. Oncol. 2020, 16, 91–102. [Google Scholar] [CrossRef] [PubMed]
  5. Sexton, R.E.; Najeeb, M.; Hallak, A.; Diab, M.; Azmi, A.S. Gastric Cancer: A Comprehensive Review of Current and Future Treatment Strategies. Cancer Metastasis Rev. 2020, 39, 1179–1203. [Google Scholar] [CrossRef]
  6. Uno, Y.; Kanda, M.; Miwa, T.; Umeda, S.; Tanaka, H.; Tanaka, C.; Kobayashi, D.; Suenaga, M.; Hattori, N.; Hayashi, M.; et al. Increased Expression of DNAJC12 Is Associated with Aggressive Phenotype of Gastric Cancer. Ann. Surg. Oncol. 2019, 26, 836–844. [Google Scholar] [CrossRef]
  7. Velu, G.; Palanichamy, V.; Rajan, A.P. Phytochemical and Pharmacological Importance of Plant Secondary Metabolites in Modern Medicine. In Bioorganic Phase in Natural Food: An Overview; Springer: Cham, Switzerland, 2018; pp. 135–156. [Google Scholar] [CrossRef]
  8. Süntar, I. Importance of Ethnopharmacological Studies in Drug Discovery: Role of Medicinal Plants. Phytochem. Rev. 2020, 19, 1199–1209. [Google Scholar] [CrossRef]
  9. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Orhan, I.E.; Banach, M.; Rollinger, J.M.; Barreca, D.; Weckwerth, W.; Bauer, R.; Bayer, E.A.; et al. Natural Products in Drug Discovery: Advances and Opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [Google Scholar] [CrossRef]
  10. Huang, M.; Lu, J.J.; Ding, J. Natural Products in Cancer Therapy: Past, Present and Future. Nat. Prod. Bioprospect. 2021, 11, 5–13. [Google Scholar] [CrossRef]
  11. Thomford, N.E.; Senthebane, D.A.; Rowe, A.; Munro, D.; Seele, P.; Maroyi, A.; Dzobo, K. Molecular Sciences Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery. Int. J. Mol. Sci 2018, 19, 1578. [Google Scholar] [CrossRef] [Green Version]
  12. Najmi, A.; Javed, S.A.; Al Bratty, M.; Alhazmi, H.A. Modern Approaches in the Discovery and Development of Plant-Based Natural Products and Their Analogues as Potential Therapeutic Agents. Molecules 2022, 27, 349. [Google Scholar] [CrossRef] [PubMed]
  13. Stojanović-Radić, Z.; Pejčić, M.; Dimitrijević, M.; Aleksić, A.; Anil Kumar, N.V.; Salehi, B.; Cho, W.C.; Sharifi-Rad, J. Piperine-A Major Principle of Black Pepper: A Review of Its Bioactivity and Studies. Appl. Sci. 2019, 9, 4270. [Google Scholar] [CrossRef] [Green Version]
  14. Haq, I.U.; Imran, M.; Nadeem, M.; Tufail, T.; Gondal, T.A.; Mubarak, M.S. Piperine: A Review of Its Biological Effects. Phyther. Res. 2021, 35, 680–700. [Google Scholar] [CrossRef] [PubMed]
  15. Subramanian, R.; Subbramaniyan, P.; Noorul Ameen, J.; Raj, V. Double Bypasses Soxhlet Apparatus for Extraction of Piperine from Piper Nigrum. Arab. J. Chem. 2016, 9, S537–S540. [Google Scholar] [CrossRef] [Green Version]
  16. Tiwari, A.; Mahadik, K.R.; Gabhe, S.Y. Piperine: A Comprehensive Review of Methods of Isolation, Purification, and Biological Properties. Med. Drug Discov. 2020, 7, 100027. [Google Scholar] [CrossRef]
  17. Rathod, S.S.; Rathod, V.K. Extraction of Piperine from Piper Longum Using Ultrasound. Ind. Crops Prod. 2014, 58, 259–264. [Google Scholar] [CrossRef]
  18. Gorgani, L.; Mohammadi, M.; Najafpour, G.D.; Nikzad, M. Sequential Microwave-Ultrasound-Assisted Extraction for Isolation of Piperine from Black Pepper (Piper nigrum L.). Food Bioprocess Technol. 2017, 10, 2199–2207. [Google Scholar] [CrossRef]
  19. Olalere, O.A.; Abdurahman, N.H.; Yunus, R.b.M.; Alara, O.R.; Akbari, S. Evaluation of Optimization Parameters in Microwave Reflux Extraction of Piperine-Oleoresin from Black Pepper (Piper nigrum). Beni-Suef Univ. J. Basic Appl. Sci. 2018, 7, 626–631. [Google Scholar] [CrossRef]
  20. Ongarora, B.C. Optimization of Piperine Extraction from Black Pepper (Piper nigrum) Using Different Solvents for Control of bedbugs. East African Agric. For. J. 2020, 84. [Google Scholar]
  21. Wu, Y.; Ma, H.; Han, Y. Solubility and Thermodynamic Properties of Piperine in (Acetone/Ethyl Acetate + Ethanol) at 278.15 K to 318.15 K and Its Correlation with the Jouyban-Acree and CNIBS/R-K Models. J. Chem. Thermodyn. 2021, 161, 106555. [Google Scholar] [CrossRef]
  22. Andrade, K.S.; Trivellin, G.; Ferreira, S.R.S. Piperine-Rich Extracts Obtained by High Pressure Methods. J. Supercrit. Fluids 2017, 128, 370–377. [Google Scholar] [CrossRef]
  23. Gorgani, L.; Mohammadi, M.; Najafpour, G.D.; Nikzad, M. Piperine—The Bioactive Compound of Black Pepper: From Isolation to Medicinal Formulations. Compr. Rev. Food Sci. Food Saf. 2017, 16, 124–140. [Google Scholar] [CrossRef] [PubMed]
  24. Tripathi, A.K.; Ray, A.K.; Mishra, S.K. Molecular and Pharmacological Aspects of Piperine as a Potential Molecule for Disease Prevention and Management: Evidence from Clinical Trials. Beni-Suef Univ. J. Basic Appl. Sci. 2022, 11, 16. [Google Scholar] [CrossRef]
  25. Jaisin, Y.; Ratanachamnong, P.; Wongsawatkul, O.; Watthammawut, A.; Malaniyom, K.; Natewong, S. Antioxidant and Anti-Inflammatory Effects of Piperine on UV-B-Irradiated Human HaCaT Keratinocyte Cells. Life Sci. 2020, 263, 118607. [Google Scholar] [CrossRef] [PubMed]
  26. Alves, F.S.; Cruz, J.N.; de Farias Ramos, I.N.; do Nascimento Brandão, D.L.; Queiroz, R.N.; da Silva, G.V.; da Silva, G.V.; Dolabela, M.F.; da Costa, M.L.; Khayat, A.S.; et al. Evaluation of Antimicrobial Activity and Cytotoxicity Effects of Extracts of Piper nigrum L. and Piperine. Separations 2023, 10, 21. [Google Scholar] [CrossRef]
  27. Corneliu Moraru, A.; Roşca, I.; Crăciun, B.; Nicolescu, A.; Chiriac, A.E.; Voicu, V. Insights of the Antimicrobial Activity of Piperine Extracted from Piper nigrum L. Farmacia 2019, 67, 6. [Google Scholar] [CrossRef]
  28. Imran, M.; Samal, M.; Qadir, A.; Ali, A.; Mir, S.R. A Critical Review on the Extraction and Pharmacotherapeutic Activity of Piperine. Polym. Med. 2022, 52, 31–36. [Google Scholar] [CrossRef]
  29. Priprem, A.; Chonpathompikunlert, P.; Sutthiparinyanont, S.; Wattanathorn, J.; Priprem, A.; Chonpathompikunlert, P.; Sutthiparinyanont, S.; Wattanathorn, J. Antidepressant and Cognitive Activities of Intranasal Piperine-Encapsulated Liposomes. Adv. Biosci. Biotechnol. 2011, 2, 108–116. [Google Scholar] [CrossRef] [Green Version]
  30. Souza, J.S.; Martins, E.P.S.; Souza, H.D.S.; De Oliveira, R.F.; Alves, F.S.; Lima, E.O.; Cordeiro, L.V.; Trindade, E.O.; Lira, B.F.; Rocha, G.B.; et al. Synthesis, Spectroscopic Characterization, DFT Calculations and Preliminary Antifungal Activity of New Piperine Derivatives. Artic. J. Braz. Chem. Soc 2021, 32, 490–502. [Google Scholar] [CrossRef]
  31. Moon, Y.S.; Choi, W.S.; Park, E.S.; Bae, I.K.; Choi, S.D.; Paek, O.; Kim, S.H.; Chun, H.S.; Lee, S.E. Antifungal and Antiaflatoxigenic Methylenedioxy-Containing Compounds and Piperine-Like Synthetic Compounds. Toxins 2016, 8, 240. [Google Scholar] [CrossRef] [Green Version]
  32. Zadorozhna, M.; Tataranni, T.; Mangieri, D. Piperine: Role in Prevention and Progression of Cancer. Mol. Biol. Rep. 2019, 46, 5617–5629. [Google Scholar] [CrossRef] [PubMed]
  33. Manayi, A.; Nabavi, S.M.; Setzer, W.N.; Jafari, S. Piperine as a Potential Anti-Cancer Agent: A Review on Preclinical Studies. Curr. Med. Chem. 2018, 25, 4918–4928. [Google Scholar] [CrossRef] [PubMed]
  34. Lee, S.H.; Kim, H.Y.; Back, S.Y.; Han, H.K. Piperine-Mediated Drug Interactions and Formulation Strategy for Piperine: Recent Advances and Future Perspectives. Expert Opin. Drug Metab. Toxicol. 2018, 14, 43–57. [Google Scholar] [CrossRef] [PubMed]
  35. Mitra, S.; Anand, U.; Jha, N.K.; Shekhawat, M.S.; Saha, S.C.; Nongdam, P.; Rengasamy, K.R.R.; Proćków, J.; Dey, A. Anticancer Applications and Pharmacological Properties of Piperidine and Piperine: A Comprehensive Review on Molecular Mechanisms and Therapeutic Perspectives. Front. Pharmacol. 2022, 12, 3549. [Google Scholar] [CrossRef] [PubMed]
  36. Mhaske, D.B.; Sreedharan, S.; Mahadik, K.R. Role of Piperine as an Effective Bioenhancer in Drug Absorption. Pharm. Anal. Acta 2018, 9, 7. [Google Scholar] [CrossRef]
  37. Srivastava, S.; Dewangan, J.; Mishra, S.; Divakar, A.; Chaturvedi, S.; Wahajuddin, M.; Kumar, S.; Rath, S.K. Piperine and Celecoxib Synergistically Inhibit Colon Cancer Cell Proliferation via Modulating Wnt/β-Catenin Signaling Pathway. Phytomedicine 2021, 84, 153484. [Google Scholar] [CrossRef]
  38. Ziegenhagen, R.; Heimberg, K.; Lampen, A.; Ildico Hirsch-Ernst, K.; Lachenmeier, W. Safety Aspects of the Use of Isolated Piperine Ingested as a Bolus. Foods 2021, 10, 2121. [Google Scholar] [CrossRef]
  39. Bolat, Z.B.; Islek, Z.; Demir, B.N.; Yilmaz, E.N.; Sahin, F.; Ucisik, M.H. Curcumin- and Piperine-Loaded Emulsomes as Combinational Treatment Approach Enhance the Anticancer Activity of Curcumin on HCT116 Colorectal Cancer Model. Front. Bioeng. Biotechnol. 2020, 8, 50. [Google Scholar] [CrossRef] [Green Version]
  40. Fattah, A.; Morovati, A.; Niknam, Z.; Mashouri, L.; Asadi, A.; Rizi, S.T.; Abbasi, M.; Shakeri, F.; Abazari, O. The Synergistic Combination of Cisplatin and Piperine Induces Apoptosis in MCF-7 Cell Line. Iran. J. Public Health 2021, 50, 1037–1047. [Google Scholar] [CrossRef]
  41. Leal, M.F.; Martins do Nascimento, J.L.; da Silva, C.E.A.; Vita Lamarão, M.F.; Calcagno, D.Q.; Khayat, A.S.; Assumpção, P.P.; Cabral, I.R.; de Arruda Cardoso Smith, M.; Burbano, R.R. Establishment and Conventional Cytogenetic Characterization of Three Gastric Cancer Cell Lines. Cancer Genet. Cytogenet. 2009, 195, 85–91. [Google Scholar] [CrossRef]
  42. Alves, F.S.; Rodrigues Do Rego, J.d.A.; Da Costa, M.L.; Lobato Da Silva, L.F.; Da Costa, R.A.; Cruz, J.N.; Brasil, D.D.S.B. Spectroscopic Methods and in Silico Analyses Using Density Functional Theory to Characterize and Identify Piperine Alkaloid Crystals Isolated from Pepper (Piper nigrum L.). J. Biomol. Struct. Dyn. 2020, 38, 2792–2799. [Google Scholar] [CrossRef] [PubMed]
  43. Sulman, L. Isolation of Piperine from Black Pepper (Piper nigrum) in the Provision of Standard Compounds for Natural Chemical Practice and Research Activities. J. Pijar MIPA 2021, 16, 683–687. [Google Scholar] [CrossRef]
  44. Pfund, L.Y.; Chamberlin, B.L.; Matzger, A.J. The Bioenhancer Piperine Is at Least Trimorphic. Cryst. Growth Des. 2015, 15, 2047–2051. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Muthuraja, A.; Subramaniyan Raja, R.; Bharath, D. Growth and Characterization of Piperine (PPN) Single Crystal Grown by Slow Evaporation Solution Growth Technique. Mater. Res. Innov. 2019, 23, 228–232. [Google Scholar] [CrossRef]
  46. Kusumorini, N.; Nugroho, A.K.; Pramono, S.; Martien, R. Development of New Isolation and Quantification Method of Piperine from White Pepper Seeds (Piper nigrum L.) Using a Validated HPLC. Indones. J. Pharm. 2021, 32, 158–165. [Google Scholar] [CrossRef]
  47. Aziz, D.M.; Hama, J.R.; Alam, S.M. Synthesising a Novel Derivatives of Piperine from Black Pepper (Piper nigrum L.). J. Food Meas. Charact. 2015, 9, 324–331. [Google Scholar] [CrossRef]
  48. Tolkatchev, D.; Elnatan, D.; Nogara, L.; Ly, T.; Naber, N.; Haak, K.; Meech, R.; Cooke, R.; Kostyukova, A.S. Piperine, an Alkaloid Inhibiting the Super-Relaxed State of Myosin, Binds to the Myosin Regulatory Light Chain. Arch. Biochem. Biophys. 2018, 659, 75. [Google Scholar] [CrossRef]
  49. Reyes-Solís, L.M.; Restrepo, J.; Sánchez, R.A. Encapsulación de La Piperine Presente En La Especie Piper Tuberculatum Utilizando Vesículas Multilamelares y Determinación de Su Poder Antioxidante. Rev. Cienc. 2018, 21, 11–28. [Google Scholar] [CrossRef] [Green Version]
  50. Lim, E.T.; Lee, J.S.; Yong, S.; Wang, T.; Zhao, Y.; Setzler, B.P.; Bahri, S.; Ambarwati, Y.; Iqbal, M.; Baihaqy, A.A. Synthesis 4-Piperoilmorpholine from Piperine. J. Phys. Conf. Ser. 2019, 1338, 012010. [Google Scholar] [CrossRef]
  51. Zarai, Z.; Boujelbene, E.; Ben Salem, N.; Gargouri, Y.; Sayari, A. Antioxidant and Antimicrobial Activities of Various Solvent Extracts, Piperine and Piperic Acid from Piper Nigrum. LWT Food Sci. Technol. 2013, 50, 634–641. [Google Scholar] [CrossRef]
  52. Perakis, F.; De Marco, L.; Shalit, A.; Tang, F.; Kann, Z.R.; Kühne, T.D.; Torre, R.; Bonn, M.; Nagata, Y. Vibrational Spectroscopy and Dynamics of Water. Chem. Rev. 2016, 116, 7590–7607. [Google Scholar] [CrossRef] [PubMed]
  53. Snyder, R.G.; Kumamoto, J.; Ibers, J.A. Vibrational Spectrum of Crystalline Potassium Hydroxide. J. Chem. Phys. 1960, 33, 1171–1177. [Google Scholar] [CrossRef]
  54. Zsila, F.; Hazai, E.; Sawyer, L. Binding of the Pepper Alkaloid Piperine to Bovine β-Lactoglobulin: Circular Dichroism Spectroscopy and Molecular Modeling Study. J. Agric. Food Chem. 2005, 53, 10179–10185. [Google Scholar] [CrossRef] [PubMed]
  55. Prabhu, G.R.D.; Kiran, C.R.; Sundaresan, A.; Mony, R.S.; Venugopalan, V.V. Process Development Studies for Recovery of Bio Active Isolates from Spent Black Pepper Generated from Ayurvedic Industry. Ind. Crops Prod. 2015, 66, 144–149. [Google Scholar] [CrossRef]
  56. Murti, Y.B.; Hartini, Y.S.; Hinrichs, W.L.J.; Frijlink, H.W.; Setyaningsih, D. UV-Vis Spectroscopy to Enable Determination of the Dissolution Behavior of Solid Dispersions Containing Curcumin and Piperine. J. Young Pharm. 2018, 11, 26–30. [Google Scholar] [CrossRef]
  57. Debnath, S.; Mishra, J. Understanding the Intrinsic Fluorescence of Piperine in Microheterogeneous Media: Partitioning and Loading Studies. New J. Chem. 2020, 44, 8317–8324. [Google Scholar] [CrossRef]
  58. Nguyen, Q.A.; Chou, W.H.; Hsieh, M.C.; Chang, C.M.; Luo, W.T.; Tai, Y.T.; Chang, W.C. Genetic Alterations in Peritoneal Metastatic Tumors Predicted the Outcomes for Hyperthermic Intraperitoneal Chemotherapy. Front. Oncol. 2023, 13, 1471. [Google Scholar] [CrossRef]
  59. Norouzi, S.; Gorgi Valokala, M.; Mosaffa, F.; Zirak, M.R.; Zamani, P.; Behravan, J. Crosstalk in Cancer Resistance and Metastasis. Crit. Rev. Oncol. Hematol. 2018, 132, 145–153. [Google Scholar] [CrossRef]
  60. Fares, J.; Fares, M.Y.; Khachfe, H.H.; Salhab, H.A.; Fares, Y. Molecular Principles of Metastasis: A Hallmark of Cancer Revisited. Signal Transduct. Target. Ther. 2020, 5, 28. [Google Scholar] [CrossRef]
  61. Yaffe, P.B.; Doucette, C.D.; Walsh, M.; Hoskin, D.W. Piperine Impairs Cell Cycle Progression and Causes Reactive Oxygen Species-Dependent Apoptosis in Rectal Cancer Cells. Exp. Mol. Pathol. 2013, 94, 109–114. [Google Scholar] [CrossRef]
  62. Cardoso, L.P.; de Sousa, S.O.; Gusson-Zanetoni, J.P.; de Melo Moreira Silva, L.L.; Frigieri, B.M.; Henrique, T.; Tajara, E.H.; Oliani, S.M.; Rodrigues-Lisoni, F.C. Piperine Reduces Neoplastic Progression in Cervical Cancer Cells by Downregulating the Cyclooxygenase 2 Pathway. Pharmaceuticals 2023, 16, 103. [Google Scholar] [CrossRef] [PubMed]
  63. Ouyang, D.Y.; Zeng, L.H.; Pan, H.; Xu, L.H.; Wang, Y.; Liu, K.P.; He, X.H. Piperine Inhibits the Proliferation of Human Prostate Cancer Cells via Induction of Cell Cycle Arrest and Autophagy. Food Chem. Toxicol. 2013, 60, 424–430. [Google Scholar] [CrossRef] [PubMed]
  64. Indrayanto, G.; Putra, G.S.; Suhud, F. Validation of In-Vitro Bioassay Methods: Application in Herbal Drug Research, 1st ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2021; Volume 46, ISBN 9780128241271. [Google Scholar]
  65. Cushnie, T.P.T.; Cushnie, B.; Echeverría, J.; Fowsantear, W.; Thammawat, S.; Dodgson, J.L.A.; Law, S.; Clow, S.M. Bioprospecting for Antibacterial Drugs: A Multidisciplinary Perspective on Natural Product Source Material, Bioassay Selection and Avoidable Pitfalls. Pharm. Res. 2020, 37, 1–24. [Google Scholar] [CrossRef] [PubMed]
  66. Lim, J.S.; Lee, D.Y.; Lim, J.H.; Oh, W.K.; Park, J.T.; Park, S.C.; Cho, K.A. Piperine: An Anticancer and Senostatic Drug. Front. Biosci. Landmark 2022, 27, 137. [Google Scholar] [CrossRef]
  67. Nakamura, H.; Maeda, H. Cancer Chemotherapy. In Fundamentals of Pharmaceutical Nanoscience; Springer: New York, NY, USA, 2023; pp. 401–427. [Google Scholar] [CrossRef]
  68. Zeien, J.; Qiu, W.; Triay, M.; Dhaibar, H.A.; Cruz-Topete, D.; Cornett, E.M.; Urits, I.; Viswanath, O.; Kaye, A.D. Clinical Implications of Chemotherapeutic Agent Organ Toxicity on Perioperative Care. Biomed. Pharmacother. 2022, 146, 112503. [Google Scholar] [CrossRef] [PubMed]
  69. Khew, C.Y.; Harikrishna, J.A.; Wee, W.Y.; Lau, E.T.; Hwang, S.S. Transcriptional Sequencing and Gene Expression Analysis of Various Genes in Fruit Development of Three Different Black Pepper (Piper nigrum L.) Varieties. Int. J. Genom. 2020, 2020, 1540915. [Google Scholar] [CrossRef]
  70. Fofaria, N.M.; Kim, S.H.; Srivastava, S.K. Piperine Causes G1 Phase Cell Cycle Arrest and Apoptosis in Melanoma Cells through Checkpoint Kinase-1 Activation. PLoS ONE 2014, 9, e94298. [Google Scholar] [CrossRef] [Green Version]
  71. Rather, R.A.; Bhagat, M. Cancer Chemoprevention and Piperine: Molecular Mechanisms and Therapeutic Opportunities. Front. Cell Dev. Biol. 2018, 6, 10. [Google Scholar] [CrossRef] [Green Version]
  72. Guo, L.; Yang, Y.; Sheng, Y.J.; Wang, J.; Ruan, S.; Han, C. Mechanism of Piperine in Affecting Apoptosis and Proliferation of Gastric Cancer Cells via ROS-Mitochondria-Associated Signalling Pathway. J. Cell. Mol. Med. 2021, 25, 9513–9522. [Google Scholar] [CrossRef]
  73. Siddiqui, S.; Ahamad, M.S.; Jafri, A.; Afzal, M.; Arshad, M. Piperine Triggers Apoptosis of Human Oral Squamous Carcinoma Through Cell Cycle Arrest and Mitochondrial Oxidative Stress. Nutr. Cancer 2017, 69, 791–799. [Google Scholar] [CrossRef]
  74. Chen, H.; Sheng, H.; Zhao, Y.; Zhu, G. Piperine Inhibits Cell Proliferation and Induces Apoptosis of Human Gastric Cancer Cells by Downregulating Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway. Med. Sci. Monit. 2020, 26, e928403. [Google Scholar] [CrossRef] [PubMed]
  75. Song, L.; Wang, Y.; Zhen, Y.; Li, D.; He, X.; Yang, H.; Zhang, H.; Liu, Q. Piperine Inhibits Colorectal Cancer Migration and Invasion by Regulating STAT3/Snail-Mediated Epithelial–Mesenchymal Transition. Biotechnol. Lett. 2020, 42, 2049–2058. [Google Scholar] [CrossRef] [PubMed]
  76. Pradeep, C.R.; Kuttan, G. Effect of Piperine on the Inhibition of Lung Metastasis Induced B16F-10 Melanoma Cells in Mice. Clin. Exp. Metastasis 2002, 19, 703–708. [Google Scholar] [CrossRef] [PubMed]
  77. Do, M.T.; Kim, H.G.; Choi, J.H.; Khanal, T.; Park, B.H.; Tran, T.P.; Jeong, T.C.; Jeong, H.G. Antitumor Efficacy of Piperine in the Treatment of Human HER2-Overexpressing Breast Cancer Cells. Food Chem. 2013, 141, 2591–2599. [Google Scholar] [CrossRef] [PubMed]
  78. Quijia, C.R.; Chorilli, M. Piperine for Treating Breast Cancer: A Review of Molecular Mechanisms, Combination with Anticancer Drugs, and Nanosystems. Phytother. Res. 2022, 36, 147–163. [Google Scholar] [CrossRef]
  79. Cheng, H.; Li, Y.; Cheng, J.; Zhang, Y.; Zhang, B. Study on the Effect and Mechanisms of Piperine against Cervical Cancer Based on Network Pharmacology and Experimental Validation. Biotechnol. Genet. Eng. Rev. 2023, 1–24. [Google Scholar] [CrossRef]
  80. Li, S.; Nguyen, T.T.; Ung, T.T.; Sah, D.K.; Park, S.Y.; Lakshmanan, V.K.; Do Jung, Y. Piperine Attenuates Lithocholic Acid-Stimulated Interleukin-8 by Suppressing Src/EGFR and Reactive Oxygen Species in Human Colorectal Cancer Cells. Antioxidants 2022, 11, 530. [Google Scholar] [CrossRef]
  81. Rehman, M.U.; Rashid, S.; Arafah, A.; Qamar, W.; Alsaffar, R.M.; Ahmad, A.; Almatroudi, N.M.; Alqahtani, S.M.A.; Rashid, S.M.; Ahmad, S.B. Piperine Regulates Nrf-2/Keap-1 Signalling and Exhibits Anticancer Effect in Experimental Colon Carcinogenesis in Wistar Rats. Biology 2020, 9, 302. [Google Scholar] [CrossRef]
  82. Araújo, T.; Khayat, A.; Quintana, L.; Calcagno, D.; Mourão, R.; Modesto, A.; Paiva, J.; Lima, A.; Moreira, F.; Oliveira, E.; et al. Piwi like RNA-Mediated Gene Silencing 1 Gene as a Possible Major Player in Gastric Cancer. World J. Gastroenterol. 2018, 24, 5338–5350. [Google Scholar] [CrossRef]
  83. Dong, P.; Xiong, Y.; Konno, Y.; Ihira, K.; Xu, D.; Kobayashi, N.; Yue, J.; Watari, H. Critical Roles of PIWIL1 in Human Tumors: Expression, Functions, Mechanisms, and Potential Clinical Implications. Front. Cell Dev. Biol. 2021, 9, 656993. [Google Scholar] [CrossRef]
  84. Buranrat, B.; Junking, M. Piperine Suppresses Growth and Migration of Human Breast Cancer Cells through Attenuation of Rac1 Expression. Asian Pac. J. Trop. Biomed. 2022, 12, 39. [Google Scholar] [CrossRef]
  85. Jeong, J.H.; Ryu, J.H.; Lee, H.J. In Vitro Inhibition of Piper Nigrum and Piperine on Growth, Migration, and Invasion of PANC-1 Human Pancreatic Cancer Cells. Nat. Prod. Commun. 2021, 16. [Google Scholar] [CrossRef]
  86. Ge, S.; Xia, X.; Ding, C.; Zhen, B.; Zhou, Q.; Feng, J.; Yuan, J.; Chen, R.; Li, Y.; Ge, Z.; et al. A Proteomic Landscape of Diffuse-Type Gastric Cancer. Nat. Commun. 2018, 9, 1012. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  87. Monster, J.L.; Kemp, L.J.S.; Gloerich, M.; van der Post, R.S. Diffuse Gastric Cancer: Emerging Mechanisms of Tumor Initiation and Progression. Biochim. Biophys. Acta Rev. Cancer 2022, 1877, 188719. [Google Scholar] [CrossRef]
  88. Tanabe, S.; Quader, S.; Ono, R.; Cabral, H.; Aoyagi, K.; Hirose, A.; Yokozaki, H.; Sasaki, H. Molecular Network Profiling in Intestinal- and Diffuse-Type Gastric Cancer. Cancers 2020, 12, 3833. [Google Scholar] [CrossRef]
  89. Gunasekaran, V.; Elangovan, K.; Niranjali Devaraj, S. Targeting Hepatocellular Carcinoma with Piperine by Radical-Mediated Mitochondrial Pathway of Apoptosis: An in Vitro and in Vivo Study. Food Chem. Toxicol. 2017, 105, 106–118. [Google Scholar] [CrossRef]
  90. Greenshields, A.L.; Doucette, C.D.; Sutton, K.M.; Madera, L.; Annan, H.; Yaffe, P.B.; Knickle, A.F.; Dong, Z.; Hoskin, D.W. Piperine Inhibits the Growth and Motility of Triple-Negative Breast Cancer Cells. Cancer Lett. 2015, 357, 129–140. [Google Scholar] [CrossRef]
  91. Afifi, N.; Barrero, C.A. Understanding Breast Cancer Aggressiveness and Its Implications in Diagnosis and Treatment. J. Clin. Med. 2023, 12, 1375. [Google Scholar] [CrossRef]
  92. Smith, B.R.; Stabile, B.E. Extreme Aggressiveness and Lethality of Gastric Adenocarcinoma in the Very Young. Arch. Surg. 2009, 144, 506–510. [Google Scholar] [CrossRef] [Green Version]
  93. Chen, D.; Xu, L.; Li, X.; Chu, Y.; Jiang, M.; Xu, B.; Zhao, M.; Wang, W.; Wang, H.; Kang, H.; et al. Enah Overexpression Is Correlated with Poor Survival and Aggressive Phenotype in Gastric Cancer. Cell Death Dis. 2018, 9, 998. [Google Scholar] [CrossRef] [Green Version]
  94. Takahashi, H.; Asaoka, M.; Yan, L.; Rashid, O.M.; Oshi, M.; Ishikawa, T.; Nagahashi, M.; Takabe, K. Biologically Aggressive Phenotype and Anti-Cancer Immunity Counterbalance in Breast Cancer with High Mutation Rate. Sci. Rep. 2020, 10, 1852. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  95. Markiewicz, A.; Nagel, A.; Szade, J.; Majewska, H.; Skokowski, J.; Seroczynska, B.; Stokowy, T.; Welnicka-Jaskiewicz, M.; Zaczek, A.J. Aggressive Phenotype of Cells Disseminated via Hematogenous and Lymphatic Route in Breast Cancer Patients. Transl. Oncol. 2018, 11, 722–731. [Google Scholar] [CrossRef] [PubMed]
  96. Joshi, S.S.; Badgwell, B.D. Current Treatment and Recent Progress in Gastric Cancer. CA Cancer J. Clin. 2021, 71, 264–279. [Google Scholar] [CrossRef]
  97. Cainap, C.; Vlad, C.; Seicean, A.; Balacescu, O.; Seicean, R.; Constantin, M.; Balacescu, L.; Crisan, O.; Marta, M.M. Gastric Cancer: Adjuvant Chemotherapy versus Chemoradiation. A Clinical Point of View. J. BUON 2019, 24, 2209–2219. [Google Scholar] [PubMed]
  98. De Oliveira, I.; Maia, A.; Fernando, J. Factors Associated with Chemotherapy Toxicity in Outpatients: A Cohort Study. Braz. J. Oncol. 2020, 16, e-20200003. [Google Scholar]
  99. Ioele, G.; Chieffallo, M.; Occhiuzzi, M.A.; De Luca, M.; Garofalo, A.; Ragno, G.; Grande, F. Anticancer Drugs: Recent Strategies to Improve Stability Profile, Pharmacokinetic and Pharmacodynamic Properties. Molecules 2022, 27, 5436. [Google Scholar] [CrossRef] [PubMed]
  100. Liu, L.; Ye, Q.; Lu, M.; Lo, Y.C.; Hsu, Y.H.; Wei, M.C.; Chen, Y.H.; Lo, S.C.; Wang, S.J.; Bain, D.J.; et al. A New Approach to Reduce Toxicities and to Improve Bioavailabilities of Platinum-Containing Anti-Cancer Nanodrugs. Sci. Rep. 2015, 5, 10881. [Google Scholar] [CrossRef] [Green Version]
  101. Mokhtari, R.B.; Homayouni, T.S.; Baluch, N.; Morgatskaya, E.; Kumar, S.; Das, B.; Yeger, H. Combination Therapy in Combating Cancer. Oncotarget 2017, 8, 38022. [Google Scholar] [CrossRef] [Green Version]
  102. Plana, D.; Palmer, A.C.; Sorger, P.K. Independent Drug Action in Combination Therapy: Implications for Precision Oncology. Cancer Discov. 2022, 12, 606–624. [Google Scholar] [CrossRef]
  103. Hegeto, L.A.; Caleffi-Ferracioli, K.R.; Nakamura-Vasconcelos, S.S.; de Almeida, A.L.; Baldin, V.P.; Nakamura, C.V.; Siqueira, V.L.D.; Scodro, R.B.L.; Cardoso, R.F. In Vitro Combinatory Activity of Piperine and Anti-Tuberculosis Drugs in Mycobacterium Tuberculosis. Tuberculosis 2018, 111, 35–40. [Google Scholar] [CrossRef]
  104. Gupta, S.K.; Bansal, P.; Bhardwaj, R.K.; Velpandian, T. Comparative Anti-Nociceptive, Anti-Inflammatory and Toxicity Profile of Nimesulide vs. Nimesulide and Piperine Combination. Pharmacol. Res. 2000, 41, 657–662. [Google Scholar] [CrossRef] [PubMed]
  105. Di, X.; Wang, X.; Liu, Y. Effect of Piperine on the Bioavailability and Pharmacokinetics of Emodin in Rats. J. Pharm. Biomed. Anal. 2015, 115, 144–149. [Google Scholar] [CrossRef] [PubMed]
  106. Yadav, S.S.; Singh, M.K.; Hussain, S.; Dwivedi, P.; Khattri, S.; Singh, K. Therapeutic Spectrum of Piperine for Clinical Practice: A Scoping Review. Crit. Rev. Food Sci. Nutr. 2022. [Google Scholar] [CrossRef] [PubMed]
  107. Kesarwani, K.; Gupta, R. Bioavailability Enhancers of Herbal Origin: An Overview. Asian Pac. J. Trop. Biomed. 2013, 3, 253. [Google Scholar] [CrossRef] [Green Version]
  108. Li, C.; Wang, Z.; Wang, Q.; Ho, R.L.K.Y.; Huang, Y.; Chow, M.S.S.; Lam, C.W.K.; Zuo, Z. Enhanced Anti-Tumor Efficacy and Mechanisms Associated with Docetaxel-Piperine Combination- in Vitro and in Vivo Investigation Using a Taxane-Resistant Prostate Cancer Model. Oncotarget 2018, 9, 3338–3352. [Google Scholar] [CrossRef] [Green Version]
  109. Xie, Z.; Wei, Y.; Xu, J.; Lei, J.; Yu, J. Alkaloids from Piper nigrum Synergistically Enhanced the Effect of Paclitaxel against Paclitaxel-Resistant Cervical Cancer Cells through the Downregulation of Mcl-1. J. Agric. Food Chem. 2019, 67, 5159–5168. [Google Scholar] [CrossRef]
  110. Burande, A.S.; Viswanadh, M.K.; Jha, A.; Mehata, A.K.; Shaik, A.; Agrawal, N.; Poddar, S.; Mahto, S.K.; Muthu, M.S. EGFR Targeted Paclitaxel and Piperine Co-Loaded Liposomes for the Treatment of Triple Negative Breast Cancer. AAPS PharmSciTech 2020, 21, 151. [Google Scholar] [CrossRef]
  111. El-Shehawy, A.A.; Elmetwalli, A.; El-Far, A.H.; Mosallam, S.A.E.R.; Salama, A.F.; Babalghith, A.O.; Mahmoud, M.A.; Mohany, H.; Gaber, M.; El-Sewedy, T. Thymoquinone, Piperine, and Sorafenib Combinations Attenuate Liver and Breast Cancers Progression: Epigenetic and Molecular Docking Approaches. BMC Complement. Med. Ther. 2023, 23, 69. [Google Scholar] [CrossRef]
  112. Cisło, M.; Filip, A.A.; Offerhaus, G.J.A.; Ciseł, B.; Rawicz-Pruszyński, K.; Skierucha, M.; Polkowski, W.P.; Cisło, M.; Filip, A.A.; Arnold Offerhaus, G.J.; et al. Distinct Molecular Subtypes of Gastric Cancer: From Laurén to Molecular Pathology. Oncotarget 2018, 9, 19427–19442. [Google Scholar] [CrossRef] [Green Version]
  113. Lauren, P. The Two Histological Main Types of Gastric Carcinoma: Diffuse and So-Called Intestinal-Type Carcinoma. Acta Pathol. Microbiol. Scand. 1965, 64, 31–49. [Google Scholar] [CrossRef]
  114. Tolosa, L.; Donato, M.T.; Gómez-Lechón, M.J. General Cytotoxicity Assessment by Means of the MTT Assay. Methods Mol. Biol. 2015, 1250, 333–348. [Google Scholar] [PubMed]
  115. Adan, A.; Kiraz, Y.; Baran, Y. Cell Proliferation and Cytotoxicity Assays. Curr. Pharm. Biotechnol. 2016, 17, 1213–1221. [Google Scholar] [CrossRef] [PubMed]
  116. Kumar, P.; Nagarajan, A.; Uchil, P.D. Analysis of Cell Viability by the MTT Assay. Cold Spring Harb. Protoc. 2018, 2018, 469–471. [Google Scholar] [CrossRef] [PubMed]
Molecules 28 05587 g001 550
Figure 1. Chemical Structure of the (2E,4E)-5-(1,3-benzodioxol-5-yl)-1-(piperidin-1-yl) penta-2,4-dien-1-one molecule (piperine). The molecule is composed of the linking between the amide and aromatic moieties via an aliphatic chain.
Figure 1. Chemical Structure of the (2E,4E)-5-(1,3-benzodioxol-5-yl)-1-(piperidin-1-yl) penta-2,4-dien-1-one molecule (piperine). The molecule is composed of the linking between the amide and aromatic moieties via an aliphatic chain.
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Figure 2. Photographs (top) and micrographs obtained by optical microscopy (bottom) of the P1 (A,D), P2 (B,E), and P3 (C,F) samples.
Figure 2. Photographs (top) and micrographs obtained by optical microscopy (bottom) of the P1 (A,D), P2 (B,E), and P3 (C,F) samples.
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Figure 3. (A) SEM micrograph and (B) EDS spectrum of P2.
Figure 3. (A) SEM micrograph and (B) EDS spectrum of P2.
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Figure 4. XRD diffractograms of (A) P1, (B) P2, and (C) P3 piperine samples.
Figure 4. XRD diffractograms of (A) P1, (B) P2, and (C) P3 piperine samples.
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Figure 5. 13C NMR (top) and 1H NMR (bottom) spectra of P1 (A,D), P2 (B,E), and P3 (C,F) piperine samples.
Figure 5. 13C NMR (top) and 1H NMR (bottom) spectra of P1 (A,D), P2 (B,E), and P3 (C,F) piperine samples.
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Figure 6. Fourier Transform Infrared spectra of P1 (green solid line), P2 (blue solid line), and P3 (red solid line) piperine samples (a) in the range 950–3800 cm−1. In (b), the region of 3100–3800 cm−1 is emphasized to track possible remnant contents of water and KOH in the solid samples.
Figure 6. Fourier Transform Infrared spectra of P1 (green solid line), P2 (blue solid line), and P3 (red solid line) piperine samples (a) in the range 950–3800 cm−1. In (b), the region of 3100–3800 cm−1 is emphasized to track possible remnant contents of water and KOH in the solid samples.
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Figure 7. (A) UV–Vis absorption and (B) steady-state PL (λexc. 352 nm) spectra of P1 (green solid line), P2 (blue solid line), and P3 (red solid line) piperine samples dissolved in P.A. dimethylsulfoxide.
Figure 7. (A) UV–Vis absorption and (B) steady-state PL (λexc. 352 nm) spectra of P1 (green solid line), P2 (blue solid line), and P3 (red solid line) piperine samples dissolved in P.A. dimethylsulfoxide.
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Figure 8. Cell viability of AGP01 cell line after 72 h of treatment with (A) P1, (B) P2, and (C) P3. The results are expressed in percentages regarding the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: ** p < 0.005, **** p < 0.0001.
Figure 8. Cell viability of AGP01 cell line after 72 h of treatment with (A) P1, (B) P2, and (C) P3. The results are expressed in percentages regarding the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: ** p < 0.005, **** p < 0.0001.
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Figure 9. Cell viability of different gastric cancer lineages: (A) AGP01 PIWIL1 KO, (B) ACP02, (C) ACP03, and (D) a non-tumor cell (VERO), after 72 h of treatment with P2. The results are expressed in percentages concerning the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: **** p < 0.0001.
Figure 9. Cell viability of different gastric cancer lineages: (A) AGP01 PIWIL1 KO, (B) ACP02, (C) ACP03, and (D) a non-tumor cell (VERO), after 72 h of treatment with P2. The results are expressed in percentages concerning the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: **** p < 0.0001.
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Figure 10. Cell viability of AGP01 cell line, after 72 h of treatment with (A) 5-FU, (B) 5-FU in combination with piperine, (C) GEM, and (D) GEM in combination with piperine. The results are expressed in percentages concerning the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: **** p < 0.0001.
Figure 10. Cell viability of AGP01 cell line, after 72 h of treatment with (A) 5-FU, (B) 5-FU in combination with piperine, (C) GEM, and (D) GEM in combination with piperine. The results are expressed in percentages concerning the untreated control. Each point is equivalent to the mean ± standard deviation of three replicates. Statistical analysis was performed with ANOVA followed by Bonferroni’s posttest. Significant differences: **** p < 0.0001.
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MDPI and ACS Style

Ramos, I.N.d.F.; da Silva, M.F.; Lopes, J.M.S.; Cruz, J.N.; Alves, F.S.; do Rego, J.d.A.R.; Costa, M.L.d.; Assumpção, P.P.d.; Barros Brasil, D.d.S.; Khayat, A.S. Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer. Molecules 2023, 28, 5587. https://doi.org/10.3390/molecules28145587

AMA Style

Ramos INdF, da Silva MF, Lopes JMS, Cruz JN, Alves FS, do Rego JdAR, Costa MLd, Assumpção PPd, Barros Brasil DdS, Khayat AS. Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer. Molecules. 2023; 28(14):5587. https://doi.org/10.3390/molecules28145587

Chicago/Turabian Style

Ramos, Ingryd Nayara de Farias, Monique Feitosa da Silva, Jefferson Marcio Sanches Lopes, Jordy Neves Cruz, Fabrine Silva Alves, José de Arimatéia Rodrigues do Rego, Marcondes Lima da Costa, Paulo Pimentel de Assumpção, Davi do Socorro Barros Brasil, and André Salim Khayat. 2023. "Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer" Molecules 28, no. 14: 5587. https://doi.org/10.3390/molecules28145587

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