Phytochemical screening and chemical variability in volatile oils of aerial parts of Morinda morindoides

Abstract Morinda morindoides is an important Liberian traditional medicine for the treatment of malaria, fever, worms etc. The plant was subjected to integrated approaches including phytochemical screening and gas chromatography mass spectrometry (GC–MS) analyses. Phytochemical investigation of the powdered plant revealed the presence of phenolics, tannins, flavonoids, saponins, terpenes, steroidal compounds and volatile oil. Steam distillation followed by GC–MS resulted in the identification of 47 volatiles in its aerial parts: 28 were in common including various bioactive volatiles. Major constituents of leaves were phytol (43.63%), palmitic acid (8.55%) and geranyl linalool (6.95%) and stem were palmitic acid (14.95%), eicosane (9.67%) and phytol (9.31%), and hence, a significant difference in the percentage composition of aerial parts was observed. To study seasonal changes, similarity analysis was carried out by calculating correlation coefficient (r) and vector angle cosine (z) that were more than 0.91 for stem-to-stem and leaf-to-leaf batches indicating considerable consistency.


Introduction
Morinda morindoides belongs to family Rubiaceae and is commonly known as Jologbo in Liberia. It exhibits a specific odour and is bitter in taste. It has been one of the famous traditional medicines employed in the treatment of malaria, worms, fever and as a laxative. Jologbo is a shrub that is commonly decocted by boiling the aerial parts (stems and leaves) with water for hours before administering it as medicine. Approximately, 4500 children in Liberia die each year by malaria as reported by Abdoulaye W. Dukule, a Liberian health ministry-physician in his article 'Life in Monrovia' (Somah 2005). One of the recent reports also demonstrated its larvicidal activity for the treatment of malaria (Chea & Kolawole 2014). In our previous studies on this plant, constituents were separated and analysed with high-performance liquid chromatography (Kiazolu et al. 2014). Different pharmacological studies have been recently carried out on other species of famous genus, Morinda that is well known to be active against different disease causing pathogens (Loonjang et al. 2015;Mandal et al. 2015). Qualitative phytochemical screening of medicinal plants has greatly contributed in the identification of many secondary metabolites responsible for the antimicrobial activity in plants (Deeni & Sadiq 2002) and hence employed as a diagnostic implement for further investigations on rarely studied Jologbo. The mechanisms of Jologbo's remedial effects are not fully understood due to the lack of having sufficient information about its chemical composition. Volatile oils may impart significant role in treating malaria and other diseases (Mota et al. 2012;Tasdemir et al. 2015). Hence, studies on phytochemical screening and essential oils may prove to discover active ingredients against various diseases.
Different seasons and climatic conditions may have a significant effect on the chemical composition of plants (Hussain et al. 2008), and hence, it is of great interest to study this effect in order to understand and estimate the efficacy of the herbal medicines during different seasons.
Previously, there has been a single report on the volatile composition of leaves of Jologbo using GC-MS (Kouame et al. 2010). To the best of our knowledge, there was no report on the volatile composition of its stem and no comparison of the volatile components in its aerial parts (stem and leaves) was drawn, what is aimed to be the major subject of this study.

Phytochemical analysis
Qualitative phytochemical analysis was performed using standard screening procedures and results are shown in Table S1. Both aerial parts revealed the presence of 10 important classes of compounds comprising monoterpenes, sesquiterpenes, diterpenes, phenylpropanoids, fatty acid, aldehydes, phenolics, aliphatic compounds, aromatic compounds and miscellaneous (containing, alcohols, thiazole and ketone, etc.) most of these are antimalarial (Liu et al. 1992;Reddy et al. 2007).

Identification of volatile constituents in aerial parts of Jologbo
The oil yields of leaf and stem of Jologbo were in the range between 0.088 and 0.095%. Pale yellow oil from the aerial parts obtained by means of steam distillation, was analysed using GC-MS analysis and confirmed by their retention indices (RI). GC-MS analysis of the volatile oil resulted to the identification of a total of 47 volatiles in the aerial parts on the basis of matching with NIST-08 library. Forty and 35 volatile compounds were identified in leaves and stems, which represent about 99.98 and 99.96% of the total volatiles found in leaves and stems, respectively. The relative per cent composition and RI of volatiles identified are summarised in Table S2. Major volatiles of leaf oil were phytol (43.63%), palmitic acid (8.55%), geranyl linallol (6.95%) and eicosane (5.62%) and stem oil were palmitic acid (14.95%), eicosane (9.67%), phytol (9.31%), 2,4-bis (1,1-dimethylethyl)-phenol (8.57%) and cedrol (7.77%). From these 47 volatiles, leaf and stem have 28 volatiles in common, whereas majority was found in leaf oil (40 volatiles) that shows a remarkable difference in the volatile composition of aerial parts which is of great consideration and according to the literature (Intisar et al. 2012) suggests the difference may lead to the difference in the activity of the plant against diseases. Further confirmation of four bioactive volatile standards, phytol, benzothiazole, α-ionone and β-ionone, were also confirmed by running pure standards under the same experimental condition as highlighted in Table S2, whereas classes of volatile constituents showing the relative per cent in leaves and stem are shown in Table 1.

Similarity characteristics of different batches of Jologbo
Chemometrics based similarity analysis provides interesting characteristic patterns among plant constituents (Liu et al. 2004) and similarity characteristics have previously been successfully employed to evaluate the informative patterns (Intisar et al. 2012;Kiazolu et al. 2014). Batches of stem-to-stem and leaves-to-leaves collected during different times to find the similarity or differences among their composition and results demonstrated high similarity throughout the spectra, peak area and peak height as shown in Table S3 and seasons do not impart much difference in the oil composition and suggests that the plant may be harvested and used in both these seasons without having significant difference in activity.

Conclusion
The phytochemical screening shows the different active classes of compounds and qualitative identification of individual active components of Jologbo highlights the important compounds present in its volatile oil. Moreover, the compositional analysis of the plant clearly showed remarkable differences in composition of stem and leaves while considerable similarity between stem-to-stem and leaf-to-leaf batches harvested in two different seasons both of what may be of utmost importance in future drug administration studies. Therefore, the investigation of the aerial parts of Jologbo as described in this paper provides the basis for further studies to fully elaborate the bioactive constituents and their possible bioactive values of the herbal medicine.

Supplementary material
experimental details related to the article are available in online only form alongside Tables S1, S2 and S3.

Disclosure statement
No potential conflict of interest was reported by the authors.