Evaluation of Ylang Ylang essential oil on alcohol induced hepatotoxicity in rats

The present study is aimed to evaluate the effect of ylang ylang essential oil in alcohol induced hepatotoxicity in rats. Alcohol was used as an inducing agent and Silymarin as a standard molecule. Body weight was measured at a 3 days interval till the twenty first day and at the end of the study, serum AST, ALT, TP, TC, TG, and TB biomarkers were estimated. Further, the liver tissue was evaluated for antioxidant enzymes namely CAT, LPO, SOD and GSH level. Liver weight and histopathology was accessed at the end. Phytocompounds were shortlisted from literature and curated databases. Probable targets of each compound were identified using the SwissTargetPrediction web server. Probable mechanisms of phytocompounds against Alcohol induced hepatotoxicity were analyzed by the STRING and KEGG pathway database. The network between compounds, targets, and pathways was generated via Cytoscape ver. 3.6.1. Docking was performed by AutoDock vina using PyRx0.8v. YYEO group showed increased BW compared to alcohol group and also reversed the increased serum AST, ALT, TP, TC, TG, and TB biomarkers and also increased the level of antioxidant enzymes compared to alcohol induced group. YYEO 400 mg/kg exhibited normal liver weight and histology compared to alcohol. Enrichment and network analysis identified YYEO 63 compounds as beneficial modulators of protein molecules associated with hepatotoxicity via modulating 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. Canangaterpene 1 was identified as a potent inhibitor of aldose reductase.


Introduction
The liver is the biggest glandular organ in the body and has maximum activities than other organs in the body. The Liver has a pivotal role in human metabolism and it produces so many actions related to haematology parameters, biochemical parameters, and enzyme-related activities (Sharma et al. 2009). As it is a major functional organ, injury or infection to this organ causes a lot of alterations in the human body.
Hepatic injury happens because of contagions, certain medications, natural and social factors, for example, liquor abuse or alcoholism, drug abuse, etc. (Ali et al. 2004).
One of the major problems associated with hepatic injury/damage is the continued consumption of alcohol by the locals as well as professionals. Alcohol misuse and reliance are turning out to be an undeniably major issue around the world because of its temperament as quite possibly the most generally psychoactive substance. Regular and exorbitant liquor utilization leads to around a 2.5 million deaths/year. Alcohol instigated liver injury is a leading main danger issue for the advancement of multiple liver sicknesses. Massive changes occur in tissue defense capacity after usage of liquor, along with modified reticuloendothelial paintings just as altered immune, platelet capacities, granulocyte, lymphocyte (McCuskey et al. 1991).
The utilization of herbs constituents for the management of liver illnesses has a long past and therapeutic herbs and their moieties are as yet utilized everywhere in the world in one form or the other for this reason. Numerous medicinal plants and their derivatives were used as hepato-protectants. The traditional plants available as hepatoprotective are Acacia catechu, Zingiber officinale, Podophyllum hexandrum, Garcinia indica, Asparagus racemosus, and many more (Amit et al. 2014). Also, numerous essential oils played a crucial role in the regulation of hepatotoxicity due to their high content of steroidal compounds.
Among the essential oils obtained from the different parts of plants, Ylang Ylang essential oil (YYEO) separated from the blossoms of Cananga odorata is prestigious for its scent and used in different makeup, massage oils, creams, fragrances, etc. It is extracted by water/water and steam distillation. Customarily YYEO is utilized in treating rheumatism, asthma, antiseptic, gout, stomach ailments, malaria, depression. The essential oil is specifically believed to be antimicrobial, antibiofilm, antiinflammatory, anti-vector, insect repellent, anti-diabetic, and anti-fertility and anti-melanogenesis, antioxidants, etc (Burdock et al. 2008). YYEO consisting of 161 bioactive constituents (reported) or more out of which eugenol, linalool, linalyl acetate, geranyl acetate, germacrene-d, beta-caryophyllene, farnesene, methyl benzoate, geraniol, and quercetin are said to be useful in hepatoprotection. However, no such scientific data are available on in silico and in vivo based hepatoprotective actions by YYEO to validate folkfore claims.
In the field of drug discovery, currently available system biology data in combination with computational or chemoinformatics approaches are being continuously applied to elucidate the molecular mechanisms of herbs/traditional medicines or polyherbal formulation. These methods help researchers to understand the complex relations between the regulation of pathways and protein molecules by phytocompounds.
Thus, based on earlier study reports as mentioned above, the present study is directed to examine the liver protection potency of YYEO by in silico tools (target identification, enrichment analysis, system pharmacology by network analysis, docking, and druggability studies), and by in vivo studies in rodents.

Dose selection for Ylang Ylang essential oil (YYEO)
YYEO was procured from the certified vendor. As reported in the earlier acute-toxicity studies showed no mortality amongst the treated animals at a dose of 2000 mg/kg (14 days). Depending onearlier studies, YYEO therapeutic dose was identified to be safe at 100, 200, and 400 mg/kg BW. Thus 1/5th, 1/10th, 1/20th of the LD 50 2000 mg/kg as mentioned above were administered orally for the proposed experiment (Mohan et al. 2002).

In-vitro antioxidant activity
The DPPH free radical scavenging assay of Ylang Ylang essential oil was performed as explained by Baba et al. (2014). Similarly, the nitrous oxide free radical scavenging assay was carried out as illustrated by Boora et al. (2014). Ascorbic acid was used as a standard. Suitable controls were taken for standard and test compounds and IC 50 value was calculated.

Animals
Male wistar rats weighing 180-220 g were used for the experiment. All the animals were housed in a hygienic and translucent polypropylenecage and randomized into six groups (N = 6 and n = 6) N = Number of groups and n = Number of animals in each group. All the animals were kept under 12/12 h natural light-dark cycle [27 °C temp. and 45-55% RH]. The overall research procedure was revised and approved by the

Experimental study design
Group I received normal food and water. Group-II (negative control) received Alcohol (40%) 3 ml/day orally q.d. up to 21 days. Group III (Positive control) received Silymarin 50 mg/kg orally and alcohol 3 ml/day p.o. q.d. up to 21 days. Group IV, V and VI (Disease induced) received alcohol (40%) 3 ml/day p.o. and Ylang Ylang essential oil 100, 200, and 400 mg/kg body weight p.o. once daily for 21 days respectively. The grouping of animals is illustrated in Table 1. At the end of the study blood was withdrawn via "retro-orbital plexus puncture" and was subjected for the biochemical estimation viz., TP, TC, AST, and ALT; whereas hepatic tissue homogenate was utilized for the evaluation of antioxidant biomarkers viz., LPO, SOD, Catalase, and GSH.

In-vivo biochemical estimations
At the end of the study, blood was collected from retroorbital plexus. The collected blood was centrifuged at 3000 rpm for 10 min to separate serum for the estimation of liver parameters such as AST/SGPT, ALT/SGOT, Total-Protein, Total Cholesterol and Total Bilirubin. All these parameters were measured by the YUCCA Kit Standard Procedures. Ohkawa et al. (1979) method was used to estimate the level of MDA in the liver tissue. To 0.2 ml of tissue homogenate 0.2 ml of SLS (8.1% w/v) was added followed by 1.5 ml of 20% CH 3 COOH and 1.5 ml TBA (0.8%). The above mixture was for 1 min and diluted up to 4 ml using DM water. Further, it was boiled at 90 °C for 1 h using the water bath. The whole mixture was cooled and 1 mL of DM water was added followed by 5 mL [n-butanol: Pyridine, 15:1 v/v) mixture and for 10 min the solution is centrifuged [rpm: 4000].

Determination of LPO
The Abs of the organic layer was noted at 532 nm. The cerebrum malondialdehyde (MDA) mg −1 of protein level was estimated and expressed in nanomoles using the formula, MDA = nmoles of MDA/mg of protein in the liver is where A = Abs, V = Mixture volume, E = 1.56 × 10 5 m/cm extinction coefficient, P = mg of protein per g of tissue.
Data represented in "nM of MDA/mg of protein". Ellman (1959) method was utilized to estimate liver tissue reduced GSH level. To 0.25 ml of tissue homogenate was added 2.5 ml sodium phosphate buffer followed by 50 µl DTNB [pH 7.0]. The above mixture was vortexed and incubated at 25-27 0 C for 2 min. Within 15 min, at 412 nm, read the mixture Abs and expressed GSH level in "µmoles/mg of tissue".

Determination of reduced GSH
where C0 is the original concentration, A is Absorbance, € is 13,600/M/cm, D is the "dilution factor".

Determination of catalase
50 µl of tissue homogenate was mixed with 1.95 ml of PBS 7.0pH and 1 ml of 0.7 mM H 2 O 2 solution. Then the absorbance was at 240 nm (Claiborne et al. 1985).

Determination of SOD
The capacity of SOD to antagonize the auto-oxidation of epinephrine to adrenochrome in the presence of alkaline pH. To the 25 µl of homogenate sample, 0.1 mM of epinephrine was added in carbonate buffer (pH 10.2). At 295 nm, the adrenochrome formation in the above mixture was measured using the ELISA plate reader. Further, the CAT and SOD levels were determined from the total protein value and expressed in U/mg of protein (Sun et al. 1978).

Histological preparations
The liver tissues from all the groups were added to 10% formalin solution and these tissues were processed and implanted in paraffin wax. Sections were cut into 6 µm thickness and added with haemotoxylin and eosin dye for low power histopathological examination at 40 × magnification.
In silico studies 1. Retrieval of bioactives from YYEO and collection of their information The reported bioactive from the YYEO were retrieved from the published literature and phytochemical databases. The chemical properties of each bioactive phytocompounds were assessed from the PubChem database viz., Canonical smiles, MW, MF, NHBA, NHBD, and logP values. The Druggability of each bioactive molecule was predicted by the molsoft online server.

Target identification (a) Targets related to Alcohol
A peer review of the literature was done to identify the specific protein molecules regulated by Alcohol in rodents and cell lines. Based on the literature, a datasheet was built for Alcohol regulated genes that are associated with hepatotoxicity. (b) Targets related to phytocompounds Canonical smiles of each compound are quired into Swis-sTargetPrediction (http:// www. swiss targe tpred iction. ch/) server to predict the probable protein molecules.
3. Targets (genes) enrichment analysis (a) Alcohol-modulated genes protein-protein interactions network was analysed by the STRING database and their molecular pathways were analysed by the KEGG pathways. Further, pathways associated with hepatotoxicity and its complications are sorted manually and a datasheet was prepared. (b) Phytocompounds regulated set of genes proteinprotein interactions network was analyzed by the STRING database and their molecular pathways were analyzed by the KEGG pathways. Further, pathways associated with hepatotoxicity and its complications are sorted manually and a datasheet was prepared.

Network construction
Based on the pathways analysis, the network between (i) Alcohol, its targets, and pathways; (ii) phytocompounds-proteins-pathways were constructed by the Cytoscape ver. 3.6.1. The edge count topological parameter was applied for assessing the degree of interaction among the multiple nodes to identify the maximum interacting protein molecules and pathways. 5. Docking studies Phytocompounds 2D and 3D structures were downloaded from the PubChem and converted 2D into 3D using Marvin sketch software. The energy minimization step for compounds was carried out using MMFF94 forcefield by open babel to minimize the clashes during docking simulation. X-ray crystallographic structure of protein molecules was retrieved from the RCSB PDB. Docking of protein-ligand was carried out by PyRx 0.8v software and their interactions were visualized by Discovery Studio Visualizer 2019.

Statistical analysis
The data were expressed in mean ± SEM/SD and assessed via "one-way ANOVA" followed by "Dunnet's multiple comparison" executing through GraphPad Prism. p < 0.05 were considered statistically significant.

DPPH 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 IC 50 value of YYEO was identified to be 221.08 µg/ml while Std molecule Ascorbic acid exhibited an IC 50 value of 84.82 µg/ml. The DPPH free radical scavenging activity of YYEO is shown in Fig. 1a.

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 IC 50 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, Alcoholinduced 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%).

Serum biochemical and enzyme estimations
The overall data is represented in Supplementary Table 2 and Fig. 3.

In-vivo antioxidant activity
The overall data is represented in Supplementary Table 3 and Figs. 4 and 5.

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.

Retrieval of bioactives from YYEO and collection of their information
About 65 bioactive phytocompounds were shortlisted fro-mYYEO 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.

Discussion
The present study was carried to explore the hepatoprotective and antioxidant efficacy of ylang ylang essential oil in drug-induced hepatotoxicity (Alcohol-induced hepatotoxicity) in Wistar rats. This study mainly includes three stages (1) in vitro, (2) in vivo followed by biochemical estimation, (3) In silico studies to probe the molecular mechanisms of YYEO.
Liver cells seem to take part in an assortment of protein metabolic functions, and alcohol causes effective hepatic harm at the doses used (Kenneth et al. 1992). The development of ROS cause oxidative stress and hepatocellular injury have been involved in alcoholic liver disease. It has been reported that Kupffer cells are the significant wellsprings of ROS during severe alcohol intake, and these initiated for . Moreover, alcohol-induced liver injury has been related to the expanded measure of lipid peroxidation (Nanjiet al. 2003). As a result, it's possible that the raised levels of marker chemicals including ALP, ALT, AST, and bilirubin resulted in the "loss of membrane structure and integrity" due to LPO process. In fact, in our investigation, YYEO supplementation Alcohol model was conceivably viable in blunting lipid peroxidation via reducing MDA level, recommending that the concentrate potentially has antioxidant prevention agent property to reduce membrane lipid peroxidation, which could be because of high level triterpenoids, and other moieties as "flavonoids, saponins glycosides, triterpenoids, and tannins" (Patel et al. 2011).
In the current study examination further we uncovered that long alcohol exposure, diminished ROS potency, viz., SOD and CAT, which in accordance with supposition recommended before by Balasubramaniyan et al. (2003) that decline in the action of antioxidants viz., "GSH, CAT, and SOD subsequent alcohol contact might be because of the harmful impacts of ROS or then again could be because of an immediate impact of acetaldehyde, produces from oxidation of alcohol (Das et al. 2005). In the current study, YYEO administration could reestablish the activity of these antioxidant prevention agents and could decrease the occurrence of free radicals and hepatic harm which may be due to the presence of bioactive moeties in YYEO mainly terpenoids and sterols that are known to possess antioxidant capacity (Gautam et al. 2007, Singh et al. 2008.
From our investigation, more prominent histopathological impacts were observed as cell changes that were noticed more in the Disease Control treated rats with a longer span of exposure in the liver. The DC group exhibited chronic damage and slight construct, and necrosis, and also this was found similar in the 100 mg/kg group with additional mild hemorrhage. However, PC exhibited reversible mechanism to NC via little degeneration and normal histology. PC and 400 mg/kg group exhibited similar effects on histology with slight degeneration followed by congestion.
Further, the study is extended to explore the molecular mechanisms behind the hepatoprotective action of YYEO. First, protein targets modulated by Alcohol were retrieved from literature and it was found that alcohol targeted 63 protein molecules. Enrichment analysis of 63 protein targets of alcohol were found to modulate 51 pathways associated/ related to the hepatotoxicity viz., Non-alcoholic fatty liver disease (NAFLD), Toll-like receptor, Adipocytokine, TNF, On the other hand, 63 bioactive molecules from YYEO were found to target 71 protein molecules. Enrichment analysis of 71 targets was found to modulate 62 pathways associated/related to the hepatotoxicity viz., 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. Majorly, among these pathways, Hepatitis B related mechanisms, EGFR tyrosine kinase inhibitor resistance, PI3K-Akt, HIF-1, Calcium, Estrogen, Relaxin, IL-17, NF-kappa B, MAPK signaling pathways were found to share with Alcohol and Paracetamol induced hepatotoxicity. These findings corroborate the in vitro and in vivo findings and provide support for the hepatoprotective action of YYEO. Further, based on the target prediction, therapeutic targets, and literature on YYEO constituents to target a protein molecule "Aldose Reductase (AR)" was chosen to identify YYEO constituent's intramolecular interactions with AR. Among 63 compounds,7 compounds viz., Benzyl cinnamate, Canangaterpene 1, Cinnamic acid, Kaempferol, Quercetin, Sulindac sulfone, and Isoeugenol were predicted to target AR. Among them, Canangaterpene 1 scored the lowest BE via forming 1 HBI and 6 NHBI. Canangaterpene 1 formed 7 interactions with active site residues. Interestingly, all 7 compounds formed interactions with active site residues, which supports their major AR inhibitory effect.

Conclusion
Our study suggested a significant protective effect of YYEO extract against Alcohol-induced hepatotoxicity and reports 400 mg/kg as a recommended dose in adult Wistar rats which brings serum and antioxidant biomarkers to normal level. Hence, YYEO could be one of the best sources of natural hepatoprotective agents. YYEO is rich in antioxidant compounds, which support the antioxidant and hepatoprotective properties Alcohol-induced model. Although, YYEO constituents are found to blunt molecular pathways dysregulated by Alcohol. Further research work on the clarification of its gene expression and individual compound action that may exhibit potent hepatoprotective property in rodents is needed to be carried out.