Gastroprotective and anti-Helicobacter pylori potentials of essential oils from the oleoresins of Araucaria bidwillii and Araucaria heterophylla

Abstract

Plant resins or oleoresins comprise a chemically complex mixture of different classes of compounds. Oleoresin of the genus Araucaria combines essential oil (EO) and resin. It possesses gastroprotective, cytotoxic, and timicrobial, antipyretic, and anti-inflammatory activities. The study aimed to investigate the EOs from the oleoresins of two Araucaria species, A. bidwillii and A. heterophylla, chemically and biologically for their gastroprotective, anti-inflammatory, antioxidant, and anti-Helicobacter pylori potentials. The chemical composition of both species cultivated in Egypt was analyzed with GC-MS and compared with those cultivated abroad using principal component analysis (PCA). There were 37 and 17 secondary metabolites identified in A. heterophylla and A. bidwillii, respectively. The EOs of both species showed a pronounced inhibitory effect on Helicobacter pylori activity in vitro. The gastroprotective effect was assessed in vivo using ethanol-induced gastric ulcer model in rats. Inflammatory cytokines, oxidative stress, and the nuclear factor-kappa B (NF-κB) biomarkers were assessed in the stomach tissues. The ulcer index and percentage of ulcer protection were determined. Stomach sections were examined histopathologically by staining with (H/E) and periodic acid Schiff (PAS). Moreover, the proliferative index was determined using the Ki-67 immunostaining. The treatment of rats with EOs (50, 100, and 200 mg/kg, orally) 1 hour prior to ethanol administration showed promising gastroprotective, anti-inflammatory, and antioxidant potentials. These findings declared the gastroprotective role played by both EOs with the superiority of A. bidwillii over A. heterophylla via modulation of oxidative stress/NF-κB/inflammatory cytokines. Their use can be recommended to protect against the recurrence of peptic ulcers.

Keywords: Anti-Helicobacter pylori activity; Anti-inflammatory effect; Antioxidant effect; Araucaria; Gastroprotective activity; Resin oil.

Fig. 1

Experimental design for evaluating the gastroprotective effect of EOs of A. heterophylla and A. bidwillii in ethanol induced peptic ulcer model in rats

Fig. 2

GC-MS chromatogram of essential oil from A. heterophylla resin collected from Egypt showing the major oil constituents; α-pinene (57.79%) followed by caryophyllene (5.4%), d-limonene (4.82%), and trans-3-caren-2-ol (4.65%)

Fig. 3

GC-MS chromatogram of essential oil from A. bidwillii resin collected from Egypt showing the major oil constituents; α-pinene (63.4%), nonane (5.21%), and trans-3-caren-2-ol (4.37%)

Fig. 4

A PCA score plot and B loading plot of GC-MS analysis of essential oil of A. heterophylla & A. bidwillii from Egypt and A. cunninghamii & A. heterophylla from India showing the variation in the composition of the oils between the Indian and the Egyptian species

Fig. 5

The reduction in the serum levels of TNF-α and IL-1β after pretreatment with Ranitidine or Essential oils of A. heterophylla (A) or A. bidiwillii (B) at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral) in ethanol induced peptic ulcer in rats. Each point represents the mean ± SD of 8 rats. Statistical significance: * is significantly different from NC GP, P < 0.05. & is significantly different from the Eth GP, P < 0.05. ^ is significantly different from Ran GP, P < 0.05. $ is significantly different from A50 treated group, P < 0.05. # is significantly different from A100 treated group, P < 0.05. @ is significantly different from A200 treated group, P < 0.05. ! is significantly different from B50 treated group, P < 0.05. ~ is significantly different from B100 treated group, P < 0.05, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post-hoc test. NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of A. heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of A. bidiwillii at a dose of 50/100/200 mg/kg

Fig. 6

A, B The macroscopic assessments of excised stomachs as well as the change percentage of ulcer area and ulcer protection after pretreatment with ranitidine or Essential oils of A. heterophylla (A) or bidiwillii (B) at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral) in ethanol induced peptic ulcer in rats. Each point represents the mean ± SD of 8 rats. Statistical significance: * is significantly different from NC GP, P < 0.05. & is significantly different from the Eth GP, P < 0.05. ^ is significantly different from Ran GP, P < 0.05. $ is significantly different from A50 treated group, P < 0.05. # is significantly different from A100 treated group, P < 0.05. @ is significantly different from A200 treated group, P < 0.05. ! is significantly different from B50 treated group, P < 0.05. ~ is significantly different from B100 treated group, P < 0.05, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post-hoc test. NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of A. heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of A. bidiwillii at a dose of 50/100/200 mg/kg

Fig. 7

The change in the stomach tissue MDA and SOD levels after pretreatment with ranitidine or essential oils of A. heterophylla (A) or A. bidiwillii (B) at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral) in ethanol-induced peptic ulcer in rats. Each point represents the mean ± SD of 8 rats. Statistical significance: * is significantly different from NC GP, P < 0.05. & is significantly different from the Eth GP, P < 0.05. ^ is significantly different from Ran GP, P < 0.05. $ is significantly different from A50 treated group, P < 0.05. # is significantly different from A100 treated group, P < 0.05. @ is significantly different from A200 treated group, P < 0.05. ! is significantly different from B50 treated group, P < 0.05. ~ is significantly different from B100 treated group, P < 0.05, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post-hoc test. NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of A. heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of A. bidiwillii at a dose of 50/100/200 mg/kg

Fig. 8

A, B The change in the stomach tissue IKB, P-IKB, N-NF-κBp65 and C-NF-κBp65 expression levels after pretreatment with Essential oils of A. heterophylla/A. bidiwillii at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral) in ethanol-induced peptic ulcer in rats. Each point represents the mean ± SD of 8 rats. Statistical significance: * is significantly different from NC GP, P < 0.05. & is significantly different from the Eth GP, P < 0.05. ^ is significantly different from Ran GP, P < 0.05. $ is significantly different from A50 treated group, P < 0.05. # is significantly different from A100 treated group, P < 0.05.@ is significantly different from A200 treated group, P < 0.05. ! is significantly different from B50 treated group, P < 0.05. ~ is significantly different from B100 treated group, P < 0.05, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post-hoc test. NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of A. heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of A. bidiwillii at a dose of 50/100/200 mg/kg

Fig. 9

The microscopic picture of H and E stained stomachs after pretreatment with Ranitidine or Essential oils of Araucaria heterophylla (A) or bidiwillii (B) at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral) in ethanol induced peptic ulcer in rats. First column refers to mucosal length affected (dashed area). Normal mucosa is seen in NC GP while a large area of mucosal necrosis. Different treated groups showed decrease of necrotic areas. (H&E, × 40) Second column illustrates depth of mucosal affection in different groups (arrows). No mucosal injury in NC GP while Eth GP shows full thickness deep mucosal injury. The depth of injury decreased in treated groups to be only superfacial erosion in Ran GP and B200 (H&E, × 100). Third column shows PAS positive mucin staining in gastric pits (red arrows). Mucin is seen in the epithelial cells of NC GP while eth Gp shows mucin depletion. Restoration of mucin was seen in treated groups. (PAS, × 200). NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of Araucaria heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of Araucaria bidiwillii at a dose of 50/100/200 mg/kg

Fig. 10

The immunohistochemical staining of Ki67 in studied groups. NC GP showing moderate nuclear positivity in neck of crypts. Eth GP showing degenerated mucosal crypts with only focal ki67 staining. After pretreatment with Ranitidine or Essential oils of Araucaria heterophylla (A) or bidiwillii (B) at 3 different doses (50, 100, 200 mg/Kg, oral) 1 h before ethanol (1 ml of 95%, oral), variable degrees of increased nuclear ki67 staining were noted indicating increased proliferation. Arrows refer to ki 67 nuclear stain. (ki 67 IHC, × 400). NC GP normal control group, Eth GP ethanol treated group, Ran GP ranitidine treated group, A50/100/200 EO of Araucaria heterophylla at a dose of 50/100/200 mg/kg, B50/100/200 EO of Araucaria bidiwillii at a dose of 50/100/200 mg/kg