Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica

Abstract

Background: The mastic gum resin has been used in traditional Kurdish medicine for treating various disorders such as topical wound and gastric ulcer. The study designed to evaluate the total polyphenol and flavonoid content, free radical scavenging activity, and anticancer effects of mastic gum resin derived from Pistacia atlantica subspecies kurdica.

Materials and methods: Folin -Ciocalteau and the aluminum chloride colorimetric assays were used to determine the total phenol and flavonoid contents in the mastic gum resin respectively. Whereas, DPPH and ABTS+ assays were used to determine the antioxidant activities of mastic gum resin. Regarding anticancer activities, the MTT assay was used to study the effect of mastic gum resin on the proliferation of various cancer cells and the morphological changes were identified after Acridine Orange/Propidium Iodide staining. Flow cytometry was applied to determine the influence of mastic gum resin on the apoptosis rate by Annexin V double staining and to investigate the influence on cell cycle progression. Caspase colorimetric assay was used to estimate the hallmark enzyme of apoptosis, and finally RNA were obtained from COLO205 cells and analyzed by qRT-PCR analyses.

Results: The MTT results showed that the mastic gum resin at concentrations from 0.01 to 100 μM induced death of cancer cells in a dose and time-dependent manner. The mastic gum resin suppressed proliferation of human cancer cells with 72 h IC₅₀ value of 15.34 ± 0.21, 11.52 ± 0.18, 8.11 ± 0.23 and 5.2 ± 0.8 μg/mL for bile duct cancer (cholangiocarcinoma) (KMBC), pancreatic carcinoma (PANC-1), gastric adenocarcinoma (CRL-1739), and colonic adenocarcinoma (COLO205) cells, respectively. Normal human colon fibroblast (CCD-18Co) cells were not adversely affected by resin treatment. Flow cytometry showed that the mastic gum resin significantly (P<0.05) arrested COLO205 cell proliferation at the G2/M phase of cell cycle. The resin caused apoptotic morphological changes in COLO205 cells. The apoptotic effect to mastic gum resin was via the mitochondrial as shown by the up-regulation of Bax, down-regulation of Bcl-2 genes, and activation of caspase-9 and -3 activities.

Conclusion: It was confirmed that the antiproliferative efficacy of the resin is positively correlated with its polyphenolic contents, suggesting a causal link related to exudate content of phenolic acid and flavonoids. The results revealed that the mastic gum resin has potential to be developed as an anticancer and antioxidant product due to its high content of polyphenol compounds.

Keywords: apoptosis; free radical scavenging; natural plant exudate; polyphenolic contents.

Figure 1

(A) Baneh or Daraban tree with clay cup for collecting resin. (B) The handmade muddy cup that was used for collecting exudate (resin). (C) Chewing gum produced from the natural MGR. Abbreviation: MGR, mastic gum resin.

Figure 2

Total phenolic and flavonoid contents of MGR from Pistacia atlantica subspecies kurdica. Each value is the average of relative SD of 3 analyses. Abbreviation: MGR, mastic gum resin.

Figure 3

Increase in radical scavenging activity of MGR: (A) DPPH and (B) ABTS+. *Significant difference from control (P < 0.05). Abbreviation: MGR, mastic gum resin.

Figure 4

(A) IC₅₀ value of 15.34 ± 0.21, 11.52 ± 0.18, 8.11 ± 0.23, and 5.2 ± 0.8 μg/mL at 72 hours of treatment with MGR for bile duct cancer (cholangiocarcinoma) (KMBC), pancreatic carcinoma (PANC-1), gastric adenocarcinoma (CRL-1739), and colonic adenocarcinoma (COLO205) cells. (B) The normal human colon fibroblasts (CCD-18Co) did not show any adverse effect after treatment with various doses of MGR. Abbreviation: MGR, mastic gum resin.

Figure 5

Confocal micrographs of AO and PI double-stained COLO205 cells. Cells were treated with IC₅₀ of MGR in a time-dependent manner. (A) Untreated cells showed normal structure without prominent apoptosis and necrosis. (B) Early apoptosis features were seen after 24 hours representing intercalated AO (bright green) among the fragmented DNA. (C) BL and nuclear margination were noticeable after 48 hours of treatment (arrows). (D) Late apoptosis was seen after 72 hours of treatment (a positive staining of orange color represents the hallmark of late apoptosis). Abbreviations: VC, viable cells; BL, blebbing; CC, chromatin condensation; MN, margination of the nucleus; EA, early apoptosis; AB, apoptotic body; LA, late apoptosis; MGR, mastic gum resin; PI, propidium iodide; AO, acridine orange.

Figure 6

Induction of apoptosis in colonic adenocarcinoma (COLO205) cells by MGR which was observed by staining with FITC-conjugated annexin V-FITC. (A1–C1) Untreated (control) COLO205 cells at 24, 48, and 72 hours. (A2–C2) COLO205 cells after treatment with MGR at 12, 24, and 48 hours. *Significant (P < 0.05) increase in apoptotic cells in the MGR-treated groups compared to untreated controls. Abbreviation: MGR, mastic gum resin.

Figure 7

The cell cycle of colonic adenocarcinoma (COLO205) cells treated with MGR after staining with PI. (A1–C1) Untreated (control) COLO205 cells at 24, 48, and 72 hours. (A2–C2) COLO205 cells treated with resin at 12, 24, and 48 hours. G0/G1, G2/M, and S are cell cycle phases, and sub-G0/G1 is the apoptotic cell population. Abbreviation: MGR, mastic gum resin.

Figure 8

Activities of caspase-3 and -9. The total cell lysates from COLO205 cells treated with MGR for 24, 48, and 72 hours were analyzed using caspase colorimetric protease assay kits. *Significant difference from control (P < 0.05). Abbreviation: MGR, mastic gum resin.

Figure 9

mRNA expression levels of Bcl-2, Bax, and Cyt-c normalized to the transcription levels of β-actin and GAPDH. The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control (P < 0.05). Abbreviations: MGR, mastic gum resin; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 10

Proposed model of MGR mechanism of action behind its antiproliferative and apoptosis effects against colon adenocarcinoma in vitro. Abbreviation: MGR, mastic gum resin.