Chemical composition and anti-angiogenic activity of the essential oil from Blumea eriantha D.C.

Abstract Blumea eriantha D.C is a weed from Asteraceae family and is reported to have anticancer activity. The essential oil from the aerial parts was extracted by steam distillation method with the yield of 0.36%. Through GC-MS analysis of the oil, seventeen compounds could be identified by comparing with linear retention indices with the library. Out of the seventeen compounds β-Caryophylline oxide was isolated by column chromatography with gradient elution and the structure was determined through FT-IR, MS, 1HNMR, 13 C NMR and DEPT. The oil was evaluated for its effect on angiogenesis using Chorioallantoic Membrane Assay (CAM Assay). The concentration dependent antiangiogenic effect was observed with IC 50 value of 19.28 ppm. Graphical Abstract


Introduction
The genus Blumea is one of the largest in the Inuleae-Inulinae, comprising approximately 80 species in tropical and sub-tropical Asia, Africa and Australia, with the highest diversity found in tropical Asia (Khair et al. 2014;Pornpongrungrueng et al. 2016).The plant is also utilized in Chinese traditional medicine in the form of cold and warm infusion for as diuretic emmenagogue and as a suderific respectively (Chen et al. 2009).Various phytoconstituents present in Blumea eriantha include essential oil, flavonoids (Datar et al. 2013) B eriantha DC showed a significant anti-microbial, anti-oxidant (Wang and Yu 2018), wound healing and anti-cancer activity on Hela and B16F10 melanoma cell lines (Pang et al. 2014).(4E,6Z)-allo-ocimene, carvotanacetone and dodecyl acetate from essential oil from B. eriantha indicated potential larvicidal activity against six mosquito species, including Zika virus vectors (liang et al. 2011).
The plants of genus Blumea are abundantly available weeds.Many activities including anti-cancer and wound healing have been reported for the same.Therefore, it was thought worthwhile to evaluate effect of the oil on angiogenesis.

Results and discussion
The area of plantation was around 1500 sq.ft.from which, total yield of the plant was roughly 150 Kg of the fresh plant.The aerial parts were harvested during their flowering seasons of November to February (Khair et al. 2014) (Figures S1 and S2).The fresh aerial parts of the selected plant species were identified and authenticated at Blatter Herbarium, St. Xavier's College, Mumbai, India.The specimen was identified as B. eriantha (matched with Blatter Herbarium specimen number Shah-1086 of G.l. Shah) belonging to the family Asteraceae.The achenes revealed to be minute angled in B. eriantha (Figure S2).The microscopic examination of the leaf epidermal surface revealed presence of anomocytic stomata and a dense covering of multicellular glandular trichomes and covering trichomes.The volatile oil of the plant may be located in the glandular trichomes.(Figure S3) The modification of the conventional distillation apparatus (Figure S4) (Gadgoli and Karve 2017) led to ease of charging and discharging of the leaves along with significant (p < 0.05) increase in the yield of volatile oil(0.7902± 0.033 gm) as compared to the conventional steam distillation apparatus (0.0.6642 ± 0.053 gm) from 500 gm of the aerial parts.
13 C DEPT analysis confirmed presence of three methyl groups at 29.91, 21.64,17.014ppm, six methylene groups at 112. 78, 39.75, 39.14,30.20,29.78, 27.The presence of caryophylline oxide was confirmed by spiking the volatile oil with the isolated caryophyllene oxide.The chromatogram obtained after spiking of the volatile oil indicated increase in the area of the peak corresponding to caryophylline (Retention time = 23.98 min) in the volatile oil.The retention time of the isolated caryophylline was found to be 23.78 min.The % content of Caryophylline oxide in the volatile oil was found to be 6.9, based on peak area (Figure S5).Also, the Molecular ion peak at 220 of the peak confirms the presence of Caryophylline oxide.
Results presented in Figure S12, indicate the mean number of branch points per square mm of Chick Chorioallantoic Membrane (CAM).Figure S11 indicates the image of the CAM for normal and treated with the volatile oil (50 ppm).Treatment with the volatile oil of B.eriantha (5 µg/ml) led to significant (p < 0.05) increase in the mean number of branch points (45.68 ± 21.31/sq mm) in the vasculature as compared to the control group (38.96 ± 14.29/sq.mm).The significant (p < 0.001) decrease in the mean number of branch points/sq.mmwas observed when the concentration of the oil was increased to 25 and 50 ppm, as compared to the control group.Further increase in the concentration did not lead to significant decrease in the mean branch points.
There was no significant change in mean number of branch points of blood vessels/ sq.mm, due to treatment of CAM with the vehicle used for the preparation of the various solutions, indicating no significant effect of vehicle on CAM.
Two standards viz.Erythropoetin, a proangiogenic agent and Adriamycin as an antiangiogenic agent were utilized for the study.Treatment with Erythropoetin (10 ppm) led to significant increase (p < 0.001) in the mean number of branch points of vessels from 38.96 to 58.97/sq.mm.Treatment with Adriamycin (10 ppm) indicated significant (p < 0.001) decrease in the mean number of branch points of vessels/sq mm from 38.96 to 19.26.
The significant reduction in the mean number of branch points of blood vessels of CAM due to treatment with the oil at the concentrations of 50 ppm indicated antiangiogenic effect with the IC 50 value of the oil of 32.6 ppm (Figure S13).The lower concentrations oil (5 ppm), exhibited pro-angiogenic activity, as the mean number of branch points of erythropoietin (10 ppm) and the volatile oil (5 ppm) were similar (Medeiros da Silva et al. 2010).The pro-angiogenic effect of erythropoetin is revealed through the significant (p < 0.001) increase in the mean number of branch points of vessels as compared to the control group.The antiangiogenic effect of the oil was found to be comparable to the anticancer drug Adriamycin (10 ppm), which is a topisomerase II inhibitor (Xu et al. 2011), as there is no significant difference between the mean number of branch points of vessels in the oil treated group as compared to the Adriamycin treated group.
Antiangiogenic effect of any agent or extract is useful for treatment of cancer, hemangioma, ophthalmic diseases, arthritis and psoriasis, while proangiogenic effect is observed to be useful in ischemia-associated diseases (Jaquet et al. 2002;Xu et al. 2011).
Caryophylline oxide is a sesqueterpene compound and is an approved naturally occuring flavorant by Food Drugs Administration (FDA) and by European Food Safety Authority.The compound is reported to be one of the natural compounds, possessing anticancer and analgesic properties (Fidyt et al. 2016).The anticancer properties of the compound have been studied in PC-3 prostate cancer and MCF-7 Breast cancer.It is reported that the methylene group and the epoxide ring of the molecule help to bind covalently with the sulfhydryl and amino group of the proteins and also bind to DNA bases thus leading to the suppression of cellular growth.The molecule is also proposed to generate Reactive Oxygen Species (ROS),in turn leading to activation of Mitogen Protein Activated Kinase (MAPK) pathway and this may be responsible for action like VEGF inhibition and thus leading to antiangiogenic activity (Brock and lee 2002;Gore and Desai 2014).B. eriantha oil indicated potent antiangiogenic activity with IC 50 value of 19.28 ppm (Figure S13).

Conclusion
From the present studies on essential oil of B. eriantha, it is concluded that the oil contains Caryophylline oxide as one of the major constituents.The oil exhibited proangiogenic effect in lower concentrations while antiangiogenic effect at higher concentrations in Chick CAM assay.