In vitro Evaluation of Cytotoxic Properties of Piperic Acid

A daily diet rich in spices may offer protection against cancer and other illnesses. This may be reason, why Indians suffer lower cases of many cancers. One of the most widely traded spices in the world, Piperine, the main alkaloid from black pepper has been shown to substantially increase the bioavailability of the nutrients in foods and supplements. Piperic acid is a chemical obtained by the hydrolysis of the alkaloid piperine from black pepper, followed by acidification of the corresponding salt. The cytotoxic effects of piperic acid are hither to unknown. In this paper, the cytotoxic effects of piperic acid in prostate cancer cells (PC-3) and breast cancer cells (MDAMB-231) were studied. The drug treatment experiments clearly indicated that maximum cytotoxicity was achieved at 48 hours and at 100μM concentration of piperic acid in both the cell lines.


INTRODUCTION
Ayurvedic medicine is a system of diagnosis and treatments that has been practiced in India for more than 2500 years. The term "ayurveda" comes from Sanskrit. It means "knowledge of life". Ayurvedic theory holds that the human body represents the entire universe in microcosmic form, and that we come to know how we function as organisms only by observing and understanding the world around us. Modern pharmaceutical research is concerned with all aspects of identifying new chemical substances with new modes of action.
The fruit of the Black Pepper plant, Black Pepper is useful both as a spice and an Ayurvedic medicine 1 . The spiciness in Black Pepper is due to the chemical Piperine. One of the most widely traded spices in the world, Piperine, the main alkaloid from black pepper has been shown to substantially increase the bioavailability of the nutrients in foods and supplements 2,3 . Piperic acid is a chemical often obtained by the base-hydrolysis of the alkaloid piperine from black pepper, followed by acidification of the corresponding salt .4,5,6,7 Piperic acid is an intermediate in the synthesis of other compounds such as piperonal, and as-such may be used to produce fragrances, perfumes flavorants and drugs as well as other useful compounds 8,9,10,11 . The structure of piperic acid was given in Figure 1.
The cytotoxic effects of piperine, an alkaloid found in black pepper, have been studied extensively 12,13,14,15 . However the cytotoxic activity of its derivative, piperic acid has not been properly investigated. The present paper is focused on the research works to fill the gap and examine the cytotoxic effects of piperic acid.

Cell lines used
PC-3 (Prostate cancer) and MDA-MB-231 (Metastatic Breast Cancer) Cell lines was obtained from NCCS (National centre for cell science), Pune, India. The PC-3 and MDA-MB -231 cells were cultured in DMEM supplemented with 10% FBS and antibiotics. The cell lines were maintained at 37°C in a 5% CO 2 incubator.

Culturing of Mononuclear cells from peripheral blood
5ml peripheral blood was collected in heparininzed blood collection tubes.
The sample was carefully added to the layer of the Ficoll Histopaque 1077 (Lymphocyte separating medium whose density is 1.077 times higher than peripheral blood) (1:1) in 15ml tubes. The tubes were centrifuged at 7000 rpm for 30 min without brake. The buffy coat was carefully aspirated and the mononuclear dense ring was collected in fresh sterile 15ml centrifuge tubes. The collected cells were washed two times using 10ml DMEM. The cell pellet was suspended DMEM enriched with 10% heat-inactivated fetal bovine serum and antibiotics. The cells were maintained in 5% CO 2 incubator at 37 0 C.

Drug treatment and Cell viability / cytotoxicity analysis using MTT assay
Mononuclear cells were obtained as mentioned previously and cultured in DMEM serum containing medium. The trypsinised cancer cells from T-25 flask were incubated in a 96 well plate and allowed to adhere to the wells overnight in CO 2 incubator. Every time 5000 cells per well were taken. Cell lines were treated with different concentrations of piperic acid (1,10,100 µM solutions). After treatment at various time intervals (24, 48 and 72 hours) , 20µl of MTT (5mg/ml in PBS) was added into each well and incubated in a CO 2 Incubator until purple precipitate was visible. Then the supernatant was discarded and 200 µl of DMSO was added to each well to dissolve formazan crystals. The absorbance was read at a wavelength of 492nm on microplate reader The following formula was used to calculate cell viability % Cell viability: (Test OD 492 / Control OD 492 ) × 100 % Cytotoxicity = 100 -% Cell viability

RESULTS AND DISCUSSIONS
MDA-MB-231, PC-3 and Mononuclear cells were cultured in 96 well plates. Once the MTT assay was conducted on the MNCs and MDA-MB-231, after the incubation period the cell viability was calculated and the following graph was obtained.
After the cells were treated with 1 micromolar concentrations of piperic acid (as shown in Table 1 and Figure 3), it was found that the cell death was much higher in the metastatic breast cancer cell lines than the mononuclear cells. This is certainly ideal as this indicates the drug can probably be safely injested into the body without causing harm to healthy cells. From the graph it was understood that piperic acid interfere with the DNA replication process hence halting the cell cycle and replication. The cytotoxic properities of 10 µM Piperic acid on MDA-MB 231 and mononuclear cells were given in Table 2 and Figure 4.
The results obtained after treatment of 10 µM piperic acid were given in Table 2 and Figure 4. In both types of cells cytotoxicity appeared to have peaked after 48 hours of incubation. This seemed to be the point where the cells had completely utilized the entire drug that was added. It is important to note that a decrease in cytotoxicity does not indicate that cell death has been reversed in any way, since these values were calculated relative to control. The cytotoxic properities of 100 µM Piperic acid on    Table 3 and Figure 5.

MDA-MB 231 and mononuclear cells were given in
The results obatined at 100 micromolars also exhibits a similar trend to the previous concentrations. The percentage of cytotoxicity was maximum at this particular concentration of piperic acid in the MDA-MB-231 cells.
Once the MTT assay was conducted on the MNCs and PC-3 cells, after the incubation period the cell viability was calculated and the following graph was obtained.
After the cells were treated with 1 micromolar concentrations of piperic acid (Table 4 and Figure 6), it was found that the cell death was much higher in the metastatic breast cancer cell lines than the mononuclear cells. This is certainly ideal as this indicates the drug can probably be safely injested into the body without causing harm to healthy cells. From the graph it was understood that piperic acid interfere with the DNA replication process hence halting the cell cycle and replication. The cytotoxic properities of 10 µM Piperic acid on PC-3 and mononuclear cells were given in Table 5 and Figure 7.
The results obtained after treatment of 10 µM piperic acid were given in Table 5 and Figure 7. In both types of cells cytotoxicity appeared to have peaked after 48 hours of incubation. This seemed to be the point where the cells had completely utilized the entire drug that was added. It is important to       note that a decrease in cytotoxicity does not indicate that cell death has been reversed in any way, since these values were calculated relative to control. The cytotoxic properities of 100 µM Piperic acid on PC-3 and mononuclear cells were given in Table 6 and Figure 8.
The results obatined at 100 µM also exhibits a similar trend to the previous concentrations. The percentage of cytotoxicity was maximum at this particular concentration of piperic acid in the PC-3 cells.
In the prostate cancer (PC-3) cells when treated with piperic acid at different time periods of incubation, it was estimated that the optimal concentration was 100 µM.

CONCLUSIONS
It is important to understand the mechanism of cell death in MDA-MB231 and PC-3 cells when treated with piperic acid. Both the cell lines were treated with piperic acid in different concentrations (1,10 and100 micromolars), at different time periods of incubation (24,48,72 hours.) The mononuclear cells were isolated from healthy human volunteers peripheral blood. These MNCs were also treated as mentioned above. From these experiments it was estimated that 100 micromolar concentration was optimal to obtain the maximum cytotoxicity in cancer cells. This concentration did not affect the cell viability of the mononuclear cells. At all the concentrations the optimal time of incubation was measured to be 48 hours. After this time period the cytotoxicity was reduced when measured at 72 hours. Perhaps the cytotoxicity would have been maintained if a second dose of the drug were administered. This decrease in cell death indicates that after the drug had been completely utilized by the cells and shown its effect, the few cells which were still alive after 48 hours of incubation began to multiply thereby decreasing the percentage of cytotoxicity.