Experimental and Computational analysis of Phytocompounds in Spathodea campanulata leaves
Priyanka Pattnayak, Pankhuri Tiwari,
Abstract: Spathodea campanulata (Fountain Tree) has several remedial uses. The bark has antiseptic properties and can be used as a laxative and its other parts like roots, seeds and flowers also have medicinal uses. The leaves of Spathodea contain iridoids, flavonoids and phenylpropanoids; classes of plant secondary metabolites. The leaves of S. campanulata is collected, dried, grinded and extracted with ethanol. The extract was analyzed by GC-MS. The peaks obtained from the chromatogram provided the names and structures of the phytocompounds through NIIST library. They are further assessed with web-enabled bioinformatics softwares such as Molinspiration, Osiris and Prediction of activity spectra of biologically active substances (PASS). The presence of various bioactive compounds and the validation of their therapeutic properties justify the use of leaves for various ailments by traditional practitioners.
Keywords: S. campanulata, GC-MS, NIIST library, Molinspiration, Osiris and PASS
Medicinal plants have been providing natural cures for centuries as they synthesize a wide variety of compounds with therapeutic properties. Their mechanism of treating human body is very much similar to the conventional drugs. The study of traditional human uses of plants is an effective way to discover future medicines. However, not much information is available on such activity of plants. Extensive studies being carried out on a daily basis which analyzes quality and effectiveness of herbal medicines.
Spathodea belongs to the family “Bignoniaceae” and is amonotypicgenus i.e. it contains a single species. The seeds are edible and used in many parts of Africa which is the native of this tree. There it is used to treat dysentery, ulcers, urinary inflammations and many more illnesses . The bark has laxative and antiseptic properties, which can be chewed and sprayed over swollen cheeks. The seeds, flowers and roots also have certain medicinal properties.
II.MATERIALS AND METHODS
A. Collection of plant materials
The leaves of Spathodea campanulata were collected from VIT Nursery, VIT University, Tamil Nadu.
B. Preparation of Sample
The leaves were shade-dried for 3 days. The leaves are then pulverized to powder with an electric grinder. The resulting 350 g of sample was stored in an air-tight container .
The sample was the extracted with 1000 ml of ethanol for 3 days. The flasks were kept at room temperature and were stirred at 1 hr interval with the help of a glass rod. The contents were filtered by Whatmann No.1 filter paper and were evaporated to dryness and concentrated by using a rotary evaporator at 75 ºC. The final residue thus obtained was then subjected to GC-MS analysis .
D. GC-MS Analysis
A Perkin Elmer GC-MS (Model Perkin Elmer Clarus 600, USA) with a VF-5 MS fused silica capillary column (30m x 0.25mm i.d, film thickness 0.25μm) was used to perform the GC-MS analyses of the sample. An electron ionization system with ionization energy of 70 eV was used for GC-MS spectroscopic detection. Pure helium gas was used as a carrier gas at a constant flow rate of 1mL/min .
E. Identification of Phytocompounds
Interpretation on mass spectrum was conducted using NIST (National Institute Standard and technology). It is a database having more than 62,000 patterns . The spectrum of the unidentified component was compared with the spectrum of the known components stored in the NIST library .
Each peak obtained from the chromatogram has a repertoire of phytocompounds, which are then analyzed using web-enabled bioinformatics software for their molecular structure, properties and their pharmacological uses.
With the help of computer program PASS, biological activity prediction of ethanolic extract of S. campanulata was obtained. PASS (Prediction of activity spectra for substances) helps in stating all the pharmacological properties of phytocompounds. PASS works on the basis of structural activity relationship (SAR) analysis . Estimates compounds action in pharmacological effects, interactions with enzymes, effect on gene expression etc. .
Molinspiration is used to test the bioavailability characteristics such as adsorption, distribution, metabolism, elimination (ADME) of the lead compounds.
The Osiris software is used to reveal toxicity, mutagenicity, drug-likeness etc. Other parameters used to calculate drug score are solubility, cLogP etc. These properties play a very important role when formulating a drug.
A. GC-MS analysis
The GC-MS analysis of phytocompounds in the leaves of Spathodea campanulata revealed the presence of several phytocomponents in the ethanol extract (Fig.1). The identification of the phytocomponents was confirmed based on the peak area, retention time and molecular formula. The phytocomponents found in the ethanol extract are shown in Table 1.
Figure 1: GC-MS chromatogram of ethanol extract of leaves of Spathodea campanulata
Table 1: Phytocomponents in ethanol extract of Spathodea campanulata by GC-MS
|S.No||RT||Name of the compounds||M. Formula||M.wt||% Peak area|
B. PASS Prediction
The predicted activity spectrum of a compound is estimates as Pa (probable activity) and Pi (probable inactivity). Compounds taken into consideration for possibility of particular pharmacological activity are the ones showing more Pa value than Pi. Squalene has a Pa of 0959 for Mucomembranous protector and 0910 for BRAF expression inhibitor. Longiverbenone has a Pa of 0810 for antineoplatic so does 1,1,3,3,5,5,7,7,9,9,11,11 DODECAMETHYL- HEXASILOXANE with a Pa of 0,889. Z,Z-6,28-HEPTATRIACTONTADIEN-2-ONE is predicted to be Antieczematic and also a Mucomembranous protector.
To qualify the compound as a drug candidate there are certain criteria. Lipophilicity (miLogP), molecular weight (MW), number of rotatable bonds (NROTB) was calculated using Molinspiration software. The percentage of absorption (%ABS) is calculated by using %ABS = 109 – (0.345 x TPSA) . Molecular polar surface area (TPSA) is a very valuable constraint for the prediction of drug transport properties. The computed molecular properties are shown in Table 2.
Table 2: Molinspiration table showing values of the parameters involving characteristics such as adsorption, distribution, metabolism, elimination (ADME) of the lead compounds
a Molecular weight.
b Molecular volume.
c Percentage absorption.
d Topological polar surface area.
e Number of rotatable bonds
g Number of hydrogen bond acceptors.
h Number of hydrogen bond donors
i Number of violations.
This software assigned each component a Drug Score by combining cLogP, Solubility and toxicity risks values. All the compounds were found to be non-mutagenic and non-tumorigenic.
The Drug scores for all the constituents fall inside the required range i.e. 0 to 1, as shown in Table 3.
Table 3: Osiris table showing different parameters such as tumerogenicity, mutagenicity, reproductive effect, irritance, solubility, cLogP, & drugscore
|S.No.||Compound Name||Mutagenic||Tumerogenic||Irritant||Reproductive Effect||cLogP||Solubility||Drug score|
Validation of plants at chemical level is an important step in the use of these plant materials for both research purposes and commercial preparations. Plants have the ability to produce a wide variety of chemical compounds that perform important biological functions. The work carried out by G. Niyonzima et al., have showed that S.campanulata stem bark extracts show remarkable hypoglycemic, anti-HIV and anticomplement activities.
As discussed by Karuppasamy Balamurugan et al., 2012; and Janakiraman N et al., 2012; several phytochemical screening studies have been performed all over the world by GC-MS and it is a valuable for tool for reliable identification of phytocompounds as it indicates the existence of many compounds with different activities and structures. Mass Spectrometer analyzes the different compounds eluted at different time and identifies the nature and structure of the compounds . In the present study, compounds have been identified from the ethanol extract of the leaves of S. campanulata. According to Subramanian Sampathkumar et al., 2013; to understand the medicinally important plant and their preparation into viable drugs the knowledge of chemical constituents is important. This helps in the isolation and characterization of the components.
It is discussed by Azhar Ariffin et al., 2014; that this tool is constructed using more than 20,000 compounds revealing more than 4000 kinds of biological activities and provides an estimated profile of compound’s action. Pa and Pi values vary from 0 to 1. Pa > Pi are said to be active . However, the PASS has some limitations as it is unable to predict the activity spectrum for new compounds. But still PASS approach is better than random speculations. According to Prasad G. Jamkhande et al., 2014; the components having more Pa value than Pi are the only constituents considered possible to have certain pharmacological activity .
In Molinspiration lipophilic character log P appears to be among the most important physicochemical parameter among relation between the structure and activity as indicated by P. Lalitha, S. Sivakamasundari, 2010. In the work of C.N.Nalini et al., 2011, Osiris was used to screen virtual compound library to select those compounds that are most likely to have high binding affinities represented as drug score. Toxicity risk signals that the drawn structure can be harmful .
Further investigation of S. campanulata may add new knowledge to the information in the traditional medical systems and may ensue to the discovery of a novel drug.
The present study results confirmed the presence of various phytoconstituents in S. campanulata in varied degree. These components were found to fulfill certain criteria for to be used as a medicine for remedial action and can be later formulated as a part of potential drug.
The authors wish to thank the lab members of VIT for their contribution.
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