Composition and in vitro cytotoxic activities of essential oil of Hedychium spicatum from different geographical regions of western Himalaya by principal components analysis

The rhizome of Hedychium spicatum has been widely used in traditional medicines. The present study deals with the evaluation of the cytotoxic potential of rhizome essential oils from four different regions of the Western Himalaya (India) along with comparative correlation analysis to characterise the bioactive cytotoxic component. The essential oils were coded as MHS-1, MHS-2, MHS-3 and MHS-4, and characterised using GC-FID and GC–MS. The main volatile compounds identified were 1,8-cineol, eudesmol, cubenol, spathulenol and α-cadinol. In vitro cytotoxic activities were assessed against human cancer cell lines such as, the lung (A549), colon (DLD-1, SW 620), breast (MCF-7, MDA-MB-231), head and neck (FaDu), and cervix (HeLa). MHS-4 is significantly active in comparison to other samples against all cancer cell lines. Sample MHS-4 has major proportion of monoterpene alcohol mainly 1,8-cineol. Principal components analysis was performed for the experimental results and all four samples were clustered according to their percentage inhibition at different doses.


Introduction
The genus Hedychium consists of more than 50 species. Hedychium spicatum is a leafy plant, up to 2 m tall, having strong aromatic horizontal rhizomes. It is commonly known as 'spiked ginger lily' and 'Kapoorkachari' (Indian trade name). The plant grows wild in the Himalayan regions q 2015 Taylor & Francis *Corresponding author. Email: drmpal.nbri@rediffmail.com of India and Nepal at altitudes of 1800 -2800 m (Kirtikar & Basu 1975;Polunin & Stainton, 1984). Their rootstocks have been widely used in traditional medicines for the treatment of skin diseases, liver complaints, asthma, and as analgesic and anti-inflammatory. The essential oil of rhizome also exhibits anthelmintic, antimicrobial and antioxidant activities (Dixit & Varma 1975;Joshi et al. 2008). Previously, a variety of terpenoids (monoterpenoids, sesquiterpenoids and diterpenoids) have been reported from H. spicatum (Sharma et al. 1976;Chopra et al. 1980;Sharma and Tandon 1983;Joshi et al. 2008;Reddy et al. 2009). Considering the commercial aspects, we report in the present communication the comparative terpenoid composition and simultaneous variations of cytotoxic activity of the rhizomes essential oils of H. spicatum collected from four different regions of the western Himalaya, India.

Chemical composition of essential oil
The essential oil yield found in all four H. spicatum fresh rhizome samples (MHS-1, MHS-2, MHS-3 and MHS-4) was 0.53%, 0.24%, 0.29% and 0.38%, respectively. These essential oil samples were analyzed using GC and GC -MS. A total of 29 chemical constituents representing 84.96 -91.33% of the oil have been identified (Table S1). The essential oil from all four rhizomes of H. spicatum was marked by the presence of sesquiterpene alcohol, monoterpene alcohol and sesquiterpene hydrocarbons. The main volatile chemical constituents identified were 1,8-cineol, hedycaryol, b-eudesmol, t-eudesmol, cubenol and a-cadinol. The essential oil samples obtained from the rhizome of H. spicatum have uniform qualitative composition, whereas they differ to a considerable level, leading to variations in the distribution pattern of components.

Statistical analysis
Principal components analysis (PCA) was performed for all experimental results ( Figure 2). All four geographically different samples were clustered together according to their percentage inhibition at different doses. This showed 100 mg/mL affects more all cancer cell lines in comparison to 50 and 25 mg/mL doses and clustered in different groups. Sample MHS-2 at 50 mg/mL dose deviates towards 25 mg/mL whereas MHS-4 at 25 mg/mL dose deviates towards 50 mg/mL dose, hence MSH-2 showed the least and MHS-4 showed the most significant cytotoxic activity.

Conclusion
It was believed that the cytotoxic activity of H. spicatum was due to the presence of sesquiterpenes (Suresh et al. 2013), but, the present study revealed that the sesquiterpenes are not solely responsible for the activity because sample MHS-2 has a major proportion of sesquiterpenes, but it is the least active against all cell lines. Moreover sample MHS-4 (Almora, Binsar Uttarakhand) has a major amount of monoterpene alcohol along with sesquiterpenes comparatively in a significant amount. Thus, there may be a synergistic effect of sesquiterpenes and monoterpene alcohols for cytotoxic potency of the oil. To the best of our knowledge this is the first report regarding an interesting cytotoxic activity of the essential oils of H. spicatum rhizomes from four different geographical regions. Furthermore, essential oil obtained from the sample MHS-4 can be selected to isolate the potent cytotoxic molecules.

Supplementary material
Experimental details relating to this article are available online alongside Tables S1 and S2. http://dx.doi.org/10.1080/14786419.2015.1049176.