Antioxidant activityDPPH free radical scavenging activity

Scavenging effect of DPPH plotted in a graph with Absorbance vs Concentration of Ylang Ylang oil and compared with Ascorbic acid (Standard antioxidant agent). The DPPH scavenging response was found to be dose-dependent. All the concentrations of Ylang Ylang oil exhibited DPPH scavenging property. The concentration of 400 and 800 µg/ml showed the highest percentage inhibition i.e., 72.61% and 77.71% respectively. On the other hand, standard drug Ascorbic Acid exhibited a percentage inhibition of 85.99% and 92.99%.The IC50 value of YYEO was identified to be 221.08 µg/ml while Std molecule Ascorbic acid exhibited an IC50 value of 84.82 µg/ml. The DPPH free radical scavenging activity of YYEO is shown in Fig. 1a.

Fig. 1

Effect of YYEO on a DPPH free radical scavenging activity b NO free radical scavenging activity

NO free radical scavenging activity

Nitrous Oxide (NO) radical scavenging assay performed for YYEO and Ascorbic acid at 50–800 µg/ml concentration and % free radical scavenging was plotted against concentrations of Ylang Ylang oil v/s absorbance. At the concentration 800 µg/ml, YYEO showed percentage inhibition of 75.14% whereas Standard Drug Ascorbic acid showed 97.84% inhibition. YYEO exhibited an IC50 value of 371.83 µg/ml whereas Standard molecule Ascorbic acid showed 133.30 µg/ml.The NO free radical scavenging activity of YYEO is shown in Fig. 1b.

Effect of YYLE on body weight

The effect of YYLE on body weight in normal, Alcohol-induced liver toxicity and YYEO treated rats were examined and % change in body weight is given in Supplementary Table 1 and Fig. 2. In normal treated rats, a gradual increase in the bodyweight is observed from day 0 to 30 i.e., 2.36% on 0th day and 21.95% (p < 0.001) on day 30th. However, in the alcohol-treated group, an increase in body weight on 0th (2.23%), 3rd day (3.59%), and 6th day (5.73%) was observed and from day 9 (5.11%) to day 30 (− 1.19%) (p < 0.001) gradual decline in body weight was observed. Further, the positive control group (Silymarin) reversed the alcohol-induced decreased body weight that showed initially a 2% increase in body weight 0th day and 17.79% (p < 0.001) on the 30th day. Similarly, YYEO 200 and 400 mg/kg exhibited gradual increase in the body weight from day 0 (3.58%, 2.72%) to day 30 (9.37%, 17.65%).

Fig. 2

Effect of YYEO on body weight. All values are expressed as Mean ± SEM where n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Normal, #p < 0.05, ###p < 0.001 when compared to DC, ΔΔΔp < 0.001 when compared to PC, @@p < 0.01, @@@p < 0.001 compared to Low Dose, ^p < 0.05, ^^^p < 0.001 compared Mid Dose

Serum biochemical and enzyme estimations

The overall data is represented in Supplementary Table 2 and Fig. 3.

Fig. 3

Effect of YYEO on a total protein b total cholesterol c AST d ALT e Bilirubin f liver weight. All values are expressed as Mean ± SEM where n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Normal, #p < 0.05, ###p < 0.001 when compared to DC, ΔΔΔp < 0.001 when compared to PC, @@p < 0.01, @@@p < 0.001 compared to Low Dose, ^p < 0.05, ^^^p < 0.001 compared Mid Dose

Total protein

Disease control (115.55 ± 4.23), Positive Control (70.38 ± 4.02), 100 mg/kg group (97.24 ± 1.51) and 200 mg/kg (82.13 ± 1.94) displayed significant (p < 0.001, p < 0.05, p < 0.001 and p < 0.001) enhancement in TP level compared to Normal (67.28 ± 1.59). In treated groups such as Positive Control, 100, 200, and 400 mg/kg (70.36 ± 3.21), DC displayed significant (p < 0.001) increase in TP level. 100 and 200 mg/kg exhibited remarkable (p < 0.001, p < 0.001) increase in TP level compared to PC. 200 mg/kg and 400 mg/kg of YYEO displayed remarkable (p < 0.001) reduction in TP level compared to 100 mg/kg (Fig. 3a).

Total cholesterol

Disease control (3.22 ± 0.04), 100 mg/kg (2.86 ± 0.03), 200 mg/kg (1.84 ± 0.03) and 400 mg/kg (1.49 ± 0.02) exhibited remarkable (p < 0.001, p < 0.001 and p < 0.05) increased TC level compared to Normal (1.44 ± 0.03). DC group exhibited remarkable (p < 0.001) increase (3.22 ± 0.04) in TC level compared to Positive Control (1.49 ± 0.01), 100 mg/kg (2.86 ± 0.03), 200 mg/kg (1.84 ± 0.03) and 400 mg/kg (1.49 ± 0.02). In contrast to Positive Control, 100 mg/kg and 200 mg/kg displayed remarkable (p < 0.001) TC level enhancement. In accordance with 100 mg/kg, 200 and 400 mg/kg resulted in significant (p < 0.001) reduction in TC level (Fig. 3b).

Aspartate aminotransferase

Disease control (379.86 ± 16.30), 100 mg/kg (319.29 ± 17.57) and 200 mg/kg (188.87 ± 7.29) exhibited remarkable (p < 0.001) raise in AST level compared to Normal (112.11 ± 6.30). In accordance with treated groups (Positive Control (125.81 ± 3.42), 100 mg/kg, 200 mg/kg and 400 mg/kg (121.87 ± 1.84), Disease control group exhibited remarkable (p < 0.001) raise in AST level. Compared to Positive Control, 100 mg/kg and 200 mg/kg group and 400 mg/kg was exhibited remarkable (p < 0.001) elevation in AST level. 200 and 400 mg/kg group exhibited remarkable (p < 0.001) decline in AST level compared to 100 mg/kg group. Remarkable (p < 0.001) decline in AST level was seen in 400 mg/kg, when compared to 200 mg/kg (Fig. 3c).

Alanine aminotransferase

Disease control (286.85 ± 6.533), Positive Control (104.34 ± 2.945), 100 mg/kg (253.93 ± 3.145), 200 mg/kg (141.76 ± 7.291) and 400 mg/kg (104.61 ± 2.911) resulted remarkable (p < 0.001) rise in ALT level on compared to Normal (89.64 ± 3.124). Compared to treated groups (Positive Control, 100, 200 and 400 mg/kg), DC group showed significant (p < 0.001) increase in ALT level. 100 and 200 mg/kg groups were showing remarkable rise in ALT level compared to Positive Control. 200 and 400 mg/kg were showing remarkable (p < 0.001) decline in ALT level compared to 100 mg/kg. A significant (p < 0.001) decrease in AST level was seen in 400 mg/kg, when compared to 200 mg/kg treated group (Fig. 3d).

Bilirubin

Disease control (3.95 ± 0.40), 100 mg/kg (3.84 ± 0.15), 200 mg/kg (2.81 ± 0.11) and 400 mg/kg (1.95 ± 0.08) showed remarkable (p < 0.001, p < 0.001 and p < 0.05) rise in bilirubin compared to Normal (1.96 ± 0.09). While, Positive Control (1.96 ± 0.09), 100, 200, and 400 mg/kg exhibited remarkable (p < 0.001) decline in bilirubin. In contrast to Positive Control, 100 and 200 mg/kg displayed remarkable (p < 0.001) bilirubin level enhancement. In accordance with 100, 200, and 400 mg/kg resulted in remarkable (p < 0.001) decline in bilirubin (Fig. 3e).

In-vivo antioxidant activity

The overall data is represented in Supplementary Table 3 and Figs. 4 and 5.

Fig. 4

Effect of YYEO on a CAT b LPO c SOD. All values are expressed as Mean ± SEM where n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001 compare to Normal, #p < 0.05, ###p < 0.001 when compared to DC, ΔΔΔp < 0.001 when compared to PC, @@ p < 0.01, @@@p < 0.001 compared to Low Dose, ^p < 0.05, ^^^p < 0.001 compared Mid Dose

Fig. 5

Effect of YYEO on GSH. All values are expressed as Mean ± SEM where n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001 compare to Normal, #p < 0.05, ###p < 0.001 when compared to DC, ΔΔΔp < 0.001 when compared to PC, @@ p < 0.01, @@@p < 0.001 compared to Low Dose, ^p < 0.05, ^^^p < 0.001 compared Mid Dose

Catalase

There is no remarkable difference shown among the control, DC and other treated groups. DC (41.67 ± 15.259), PositiveControl (54.49 ± 12.275), 100 mg/kg (38.46 ± 7.402), 200 mg/kg (44.87 ± 9.065) and 400 mg/kg (51.28 ± 9.065) groups were exhibited decline in CAT level compared to Normal (54.49 ± 8.276). In contrast to treated groups (Positive Control 100, 200, and 400 mg/kg), DC exhibited decline in CAT level (Fig. 4a).

Lipid peroxidation

DC (49.68 ± 3.205) exhibited remarkable (p < 0.001) rise in LPO level on comparing to Normal Group (20.83 ± 6.137). Treated groups such as Positive Control (25.64 ± 5.234), 100 mg/kg (35.26 ± 3.701), 200 mg/kg (30.45 ± 6.137) and 400 mg/kg (20.83 ± 6.137) exhibited remarkable (p < 0.001, p < 0.05, p < 0.001, p < 0.001) decline in LPO level compared to Disease control. On comparing with Positive Control group, 100 mg/kg and 200 mg/kg groups exhibited decline in LPO level. However, 400 mg/kg group exhibited remarkable (p < 0.01, p < 0.05) decline in LPO level compared to 100 mg/kg and 200 mg/kg group respectively (Fig. 4b).

Superoxide dismutase

There is no remarkable difference shown between the control, DC, and all the treated groups. DC (70.03 ± 109.065) exhibited a decline in SOD level compared to the Normal Group (243.86 ± 49.503). Treated groups such as Positive Control (99.71 ± 165.819), 200 mg/kg (144.50 ± 121.465) and 400 mg/kg (141.55 ± 339.445) exhibited decline in SOD level compared to DC. 200, and 400 mg/kg group exhibited decline in SOD level compared to 100 mg/kg group (Fig. 4c).

Glutathione

DC (0.45 ± 0.104), Positive Control (1.26 ± 0.027), 100 mg/kg (1.23 ± 0.025), and 200 mg/kg (0.75 ± 0.010) exhibited remarkable (p < 0.001, p < 0.05, p < 0.01, p < 0.001) decline in GSH level compared to Normal Group (1.41 ± 0.073). Treated groups viz., Positive Control, 100, 200, and 400 mg/kg (1.30 ± 0.046) exhibited remarkable (p < 0.001) rise in GSH level compared to DC. When compared to PC, 200 mg/kg exhibited remarkable (p < 0.001) reduction in GSH level. 200 mg/kg exhibited remarkable (p < 0.001) reduction in GSH level compared to 100 mg/kg group. 400 mg/kg exhibited remarkable (p < 0.001) elevation in GSH level compared to 200 mg/kg (Fig. 5).

Effect on liver weight

Disease control (0.0569 ± 0.00163), 100 mg/kg (0.0467 ± 0.00163) and 200 mg/kg (0.0422 ± 0.00147) exhibited remarkable (p < 0.001, p < 0.001 and p < 0.05) increase in liver weight compared to Normal (0.0328 ± 0.00194). Likewise, DC group also saw a significant (p < 0.001) increase in liver weight compared to Positive Control (0.0318 ± 0.00160), 100, 200 and 400 mg/kg (0.0325 ± 0.00207). In contrast to Positive Control, both 100 and 200 mg/kg displayed remarkable (p < 0.001) liver weight enhancement. In accordance with 100 mg/kg, 200 mg/kg and 400 mg/kg resulted in significant (p < 0.001, p < 0.01) reduction in Liver weight (Supplementary Table 4, Fig. 3f).

Histopathology

Normal group rat’s histopathology examination displayed a normal histology at 40 × magnification while Alcohol treated rats group showed a-focal collection of lymphocytes around hepatocytes within the lobule, b-spotty necrosis, c-lymphocytic infiltration around portal tract, and micro vesicular steatosis. Silymarin group showed a spotty (minute) necrosis in hepatocytes. While rats treated with YYEO (100-400 mg/kg) showed a reduction in the lymphocytic infiltration around the portal tract and showed mild necrosis. The histopathology results have been summarised in Fig. 6.

Fig. 6

Histopathology of the Liver for Alcohol Induced Animal. a Normal group, b Disease Control, c Standard, d YYEO Low Dose, e YYEO Mid Dose and f YYEO High Dose

In silico studiesRetrieval of bioactives from YYEO and collection of their information

About 65 bioactive phytocompounds were shortlisted fromYYEO and their druggability w.r.t. Lipinski rule of five was summarized in Supplementary Table 5.

Targets related to Alcohol and Phytocompounds

Alcohol: Based on the literature, Alcohol was found to target 63 protein molecules to cause hepatotoxicity.

Phytocompounds: Based on target prediction by Swiss Target Prediction 65 phytocompounds from YYEO were predicted to modulate 71 protein molecules.

Targets (genes) enrichment and network analysisAlcohol

The enrichment analysis of 63 protein molecules targeted by Alcohol was found to modulate 182 molecular pathways. Among 182 pathways, 51 were associated/related to hepatotoxicity (Supplementary Table 6). NAFLD, Toll-like receptor, Adipocytokine, TNF, Sphingolipid, FoxO, AMPK, Relaxin, MAPK, NF-kappa B, HIF-1, Fc epsilon RI, IL-17, VEGF, T cell receptor, NOD-like receptor, mTOR, PI3K-Akt signaling pathway, etc. were found to score the least FDR value and significantly modulated by Alcohol. Figure 7 represents the alcohol-target-pathway network.

Fig. 7

Network representation of Alcohol, regulated genes, and their molecular pathways

Phytocompounds

The enrichment analysis of 71 protein molecules targeted by 63 phytocompounds were found to modulate 169 molecular pathways. Among 169 pathways, 62 were associated/related to hepatotoxicity (Supplementary Table 7). HIF-1 signaling pathway, Hepatitis B related mechanisms, EGFR tyrosine kinase inhibitor resistance, PI3K-Akt, Calcium, Estrogen, Relaxin, IL-17, NF-kappa B, TNF, MAPK signaling pathways, etc. were found to score the least FDR. Figure 8 represents the phytocompounds-protein targets andpathways network.

Fig. 8

Network representation of YYEO phytocompounds, probable protein molecules, and their molecular pathways

Docking studies

Based on the target prediction, therapeutic targets, and literature on YYEO constituents effect protein target “Aldose Reductase (AR)” was chosen to identify YYEO constituent’s interactions with AR. Among 63 compounds, 7 compounds viz., Benzyl cinnamate, Canangaterpene 1, Cinnamic acid, Kaempferol, Quercetin, and Isoeugenol were predicted to target AR. Sulindac sulfone, a known standard aldose reductase inhibitor was also docked with aldose reductase enzyme for comparison purpose. Among them, Canangaterpene 1 scored the lowest BE of—10 kcal/mol via forming 1 HBI i.e. His110, and 6 NHBI i.e., Tyr209, Trp20 (2), Cys298, Val47, Ile260. Canangaterpene 1 formed 7 interactions with active site residues. Interestingly, all 7 compounds formed interactions with active site residues (supplementary Table 8). Figure 9 represents the interaction of phytocompounds withAR.

Fig. 9

2D and 3D representation of interaction of aldose reductase with a Quercetin, b Kaempferol c Sulindac sulfone. *Sulindac sulfone is a standard aldose reductase inhibitor molecule