Anti-inflammatory and neuroprotective activity of Viphyllin a standardized extract of β-caryophyllene from black pepper (Piper Nigrum L) and its associated mechanisms in mouse macrophage cells and Human Neuroblastoma SH-SY5Y cells

Oxidative stress breeds various chronic lifestyle ailments including inflammatory conditions and neurodegenerative diseases. β-caryophyllene natural bicyclic sesquiterpene, obtained from various plants sources found to be effective against inflammation and neuroprotection. In this study, we have evaluated the protective effect of Viphyllin, a standardized extract of β-caryophyllene from black pepper against inflammation induced by lipopolysaccharide in RAW264.7 macrophage cells and mechanisms involved in hydrogen peroxide (H2O2)-challenged oxidative stress in human neuroblastoma SH-SY5Y cells. Viphyllin demonstrated the anti-inflammatory activity by subsiding the release of the pro-inflammatory intermediaries like NO, cytokines, interleukins, and protein expression levels of cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS). In addition, Viphyllin suppressed the extracellular signal-regulated kinase (ERK). c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. On the other hand, Viphyllin showed neuroprotective effect against neuronal oxidative damage caused by H2O2. Viphyllin lessened the expression B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2-associated X protein (BAX), cleaved caspase-9, and PARP-1 proteins associated with apoptosis. Our results indicate that Viphyllin ameliorated LPS-mediated inflammation in macrophages by regulating inflammation and Viphyllin exerted remarkable anti apoptotic effect against neuronal damage challenged by H2O2. Altogether, Viphyllin could be potential functional food ingredient for inflammation and neurodegenerative diseases.


Introduction.
Pharmacological properties of medicinal plants are primarily due to the presence of secondary metabolites like polyphenols, polyphenols, steroids, flavonoids, alkaloids and terpenoids [1,2]. Terpenoids have been studied as an alternative to treat different disease conditions. Certain terpenoids for instance Taxol and Artemisinin has been using as anticancer and antimalarial drug, respectively. Terpenoids play diverse role in the field of food, drugs, and cosmetics, studies show the relationship of terpenoid consumption with the reduction of inflammation [3,4]. β-caryophyllene is a "natural bicyclic sesquiterpene hydrocarbon" it is most abundant constituent exist in many plant-originated essential oils, like as lemon balm, cloves, Cannabis sativa and black pepper [5,6]. Recent studies are reported on significant biological activities of β-caryophyllene against inflammation, cancer and chronic pain and chemo sensitizing properties for chemotherapies like oxaliplatin, doxorubicin sorafenib, and 5-fluorouracil. On the other hand, β-caryophyllene is a plant (or food) -derived cannabinoid ligand, and it is also reported that it functions as a selective agonist of cannabinoid receptor 2 (CB2). It is thought that CB (2) receptor activation is suitable medicinal approach for the treatment of inflammation related disorders. United States Food and Drug Administration considers β-caryophyllene is a dietary phyto cannabinoids and has been included in the list of "generally regarded as safe" ingredients for dietary use and [7,8].
In the current investigation, we have enriched and systematized the extract in a manner that is not less than 30% β-caryophyllene and termed it as Viphyllin, subsequently investigated for anti-inflammatory activity in LPS induced mouse macrophage cells and neuroprotective property against oxidative stress induced by H 2 O 2 . To the best of our knowledge, this is the very first study revealing that a standardized extract of beta caryophyllene (Viphyllin) from black pepper demonstrated an anti-inflammatory and neuroprotective activity.

Sample preparation
Viphyllin a standardized extract of β-caryophyllene from black pepper (Piper Nigrum L) was collected from the Department of Phytochemistry, Vidya Herbs Pvt. Ltd. (Bangalore, Karnataka, India), and dissolved in cell culture media (DMEM) at the appropriate concentrations. (Supplementary figure S1) Cell culture RAW 264.7 Murine macrophage and Human neuroblastoma SH-SY5Y cells were obtained through NCCS (Pune, India), RAW264.7 cells were cultured in DMEM media containing 10% foetal bovine serum and antibiotic solution in a humidified 5% CO 2 incubator (Thermo Fisher Scientific). The cell culture medium was replaced with fresh DMEM media every 2 to 3 days, once cells reached 80% confluence, the cells were subcultured. SH-SY5Y cells were grown in DMEM and F12 nutrient mix subsidized with FBS (10%) and antibiotic solution respectively at 37 °C in the CO 2 incubator.

Cell viability assay
MTT assay was adopted to check the influence of Viphyllin on the viability of RAW 264.7 cells. Briefly, cell suspensions were seeded at density of 1×10 4 cells/well in 96-well plates.
After 18 h of incubation, the cells were treated with the different concentrations(5-50μg/mL) of Viphyllin, further incubated for 24 h. The supernatant was discarded and 100 μL of MTT solution (0.5 mg/mL) was replaced and cells were incubated for an extra 2-4hrs, subsequently 100 μL of MTT stop solution (DMSO) was added to wells to dissolve the formazan crystal.
The absorbance was read at 550 nm using a microplate reader (Tecan Infinite M200 Pro).
Likewise, cell viability assay was conducted on SHSY5Y cells. The preventive effect of Viphyllin versus H 2 O 2 -elicited neurotoxicity was investigated by incubating cells with 12 and 24µg/mL of Viphyllin for 6 h and then treated them with 500μM H 2 O 2 for 24 h. Cell viability of cells were determined using the MTT assay as described above.

Determination of NO
RAW macrophages were set at 4×10 4 cells/well in 96-well plates for 18 h. Then the cells were incubated with different concentrations (5-50μg/mL) of Viphyllin before they were stimulated with 1μg/ml lipopolysaccharide. After completion of 24h incubation, using the cell culture supernatant NO content was measured by Griess reagent.

Evaluation of cytokine secretion level in macrophages
The secretion level of PGE-2, TNF-α, IL-6 and IL-1β, inflammatory cytokines levels were measured by the ELISA using in the cell culture supernatant of RAW 264.7 macrophages according to manufacturer's protocol (Krishgen Biosystems, Mumbai, India).

Lactate dehydrogenase (LDH) assay
The assay was performed by employing an LDH cytotoxicity detection kit (Sigma aldrich).
SH-SY5Y cells were added at the density of 2.5×10 4 cells/ mL in 96-well. Then the cells were treated with Viphyllin at different concentrations (5-50μg/mL) for 24h. with and without H 2 O 2 . The amount of LDH release was determined by using a microplate reader (Tecan Infinite M200 Pro) at 490 nm.

Western Blot Analysis
RAW macrophages were incubated overnight and subjected to two different concentrations of Viphyllin 12 and 24µg/mL prior to LPS (1µg/mL) treatment. Cells were collected by trypsinization after 24 h incubation and were lysed with 100µL of radioimmunoprecipitation (RIPA) lysis buffer. Total protein concentration was measured using the Bradford reagent (Bio-Rad). Equivalent amounts of protein (30µg) were separated by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk solution (and incubated with 1:1000 diluted primary antibodies (cox2. iNOS, p-ERK, p-JNK, p-p38 and β-actin) at 4 ο C for overnight, and subsequently probed with specific secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotech, Dallas, TX, USA) and bands were visualized using ECL reagents, and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Similarly, western blot experiment was conducted in H 2 O 2 induced oxidative damage in SH-SY5Y cells using PARP1, caspase 9, Bcl-2 and BAX primary antibodies.

Statistical analysis
One -way ANOVA and Dunnett's multiple comparisons test has been used to determine the statistical significance of differences among control and treated groups. Experiments were carried out in triplicate and all the values that were expressed as a mean ± standard error.

Cell viability
We investigated the cytotoxic effect of Viphyllin in RAW264.7 murine macrophages. As shown in Figure 1A. Viphyllin had no cytotoxic effect on macrophages up to the dosage of Hence, we concluded that concentrations up to 30 μg/ml of the Viphyllin could be safe. Thus, in the subsequent experiments, RAW274.7 cells were treated with nontoxic concentrations of Viphyllin. The morphology of LPS elicited cells had an uneven shape and spreading compared to untreated cells, whereas Viphyllin treated cells offer protection against LPS treatment by maintaining normal shape of cells ( Figure 1B).  Figure 5D).

Effects of Viphyllin on apoptosis-related protein expressions in H 2 O 2 -induced SH-SY5Y cells
It has been reported that accumulation of H 2 O 2 causes apoptosis, inflammation and necrosis in the brain is well known in neurodegenerative disease.

Discussion
Persistent inflammation has a extensive role in the pathogenesis of various chronic diseases like diabetes, obesity, cardiovascular and neurological disorders [9][10][11]. Approaches to aim for inflammation has become an attractive approach to defend inflammatory-related ailments [12,13]. Over the last few years, bioactive constituents or standardized extracts obtained from plant sources still play a significant role in the treatment of many ailments without any adverse effect [14]. Macrophages are crucial participants in the immune response to foreign intruders such as infectious microorganisms. lipopolysaccharide (LPS) is a well-known prototypical endotoxin may directly activate macrophages. Plethora of measures have recommended that activated macrophage produce a greater amount of various proinflammatory intermediaries like NO and PGE2, also proinflammatory cytokines for example TNF-α, and interleukins. All these elements stimulate the development of inflammation and worsen the inflammation over the harmonious interaction with further inflammatory intermediaries and there are reports that in variety of inflammatory ailments including asthma and colitis the expression levels of PGE2 and NO is elevated [15][16][17]. Our study revealed that Viphyllin efficiently inhibited the secretion of PGE2 and NO, and proinflammatory cytokines in LPS-challenged macrophages at safer dose, our findings are consistent with earlier published studies [18,19]. COX-2. is an important target for the design of drugs against inflammation, particularly NSAIDs. Furthermore, iNOS and COX-2 enzymes play crucial role in the generation of NO and PGE2, Viphyllin significantly subsided the iNOS and COX-2 expression in LPS stimulated macrophages. Our results are consistent with the previous published reports [20,21].
The MAPK signalling cascade plays a key role in the initiation of response to inflammation. ERK, JNK, and p38 participate in MAPK signalling pathways, which gets activated by TNFα and interleukins [22]. Earlier studies have shown that LPS or TNF activates MAPKs which are involved in acute lung injury. It has been reported that when MAPK gets activated phosphorylated ERK, p38, and JNK are moved to the nucleus and stimulates response to inflammation by inducing expression of proper gene of interest [23,24]. Pre-treatment of Viphyllin inhibited MAPK signalling by subsiding the phosphorylation of ERK, JNK, and p38 in LPS induced macrophages. It is evident from the study that inhibition of MAPK pathway is a potential mechanism by which Viphyllin wields anti-inflammatory effects.
These findings are consistent with those earlier studies [25,26].
Lately, several findings have found neurodegenerative diseases caused by stress and has been extensively associated in neuronal cell death. Previous studies have reported that H 2 O 2 accumulation has been well studied in neurodegenerative disease. Human neuroblastoma SH-SY5Y cells are very well-regarded cellular tool for research on H 2 O 2 -stimulated oxidative stress [27][28][29] . In our recent study we have explored standardized β-caryophyllene extract improves cognitive function in scopolamine-induced amnesia model [30]. In current study, we have studied the protective effect of Viphyllin against H 2 O 2 -provoked apoptotic events in SH-SY5Y cells. It has been showed that H 2 O 2 stimulated oxidative damage dramatically declines viability of SH-SY5Y cells [31]. Our study also showed decline of cell viability in the presence of H 2 O 2 in SH-SY5Y cells. Interestingly we have noticed viability of cells enhanced dose dependently by the treatment of Viphyllin. It has been well established that damage to cellular membranes releases LDH into the cell culture supernatant is important marker of cell death [32]. From our data it is noticeably clear that elevated level of LDH was found in the culture medium after H 2 O 2 -induction in SH-SY5Y cells. Treatment with the Viphyllin markedly lower the LDH in the SH-SY5Y cell culture medium subjected to H 2 O 2 .
Hence, we reaffirmed the defensive effects of Viphyllin in H 2 O 2 -elicited SH-SY5Y cells. Our outcomes are constant with previously published study [33]. It has been reported that toxic effect on SHSY-5Y cell is caused by H 2 O 2 is linked with mitochondria-associated apoptotic events and it is considered as one of the valuable tools in neurodegenerative diseases [34].
Cytochrome release occurs when elevated levels of ROS penetrate mitochondrial membrane which leads to the activation of caspase -9 and PARP-1 and gradually induces cell death [35].
Our results demonstrated that markedly elevated proteolytic cleavage of caspase-9 and PARP in H 2 O 2 subjected SHSY-5Y cells. However, Viphyllin protects neuronal cells against H 2 O 2 stimulated oxidative stress by lessening the expression of caspase-9 and PARP1. Furthermore, mitochondrial membrane injury leads to neuronal apoptosis by up-regulation of Bcl-2 and down-regulation of Bax, which leads to inhibition of caspase activation [36]. In current study, Viphyllin treatment lowered Bax level and enhanced Bcl-2 level. Hence, these outcomes demonstrated that Viphyllin along with other active constituents from black pepper offered preventive effects against oxidative damage, our results were consistent with other published studies [37,38]. In conclusion, our current unearthing demonstrated that the Viphyllin standardized extract containing β-caryophyllene from black pepper exert an antiinflammatory function via the downregulation COX-2, iNOS and of the MAPK pathway in LPS challenged macrophages on the other hand it also offers neuroprotective effect. This investigation may suggest Viphyllin is encouraging bioactive food ingredients. However, imminent research is essential relating to the other controlling mechanism of Viphyllin.