Determination of metabolites products by Cassia angustifolia and evaluate antimicobial activity

Phytochemicals are chemical compounds often referred to as secondary metabolites. Forty four bioactive phytochemical compounds were identified in the methanolic leaves extract of Cassia angustifolia. The identification of phytochemical compounds is based on the peak area, retention time molecular weight and molecular formula. Gas chromatography-mass spectrometry (GC-MS) analysis of C. angustifolia revealed the existence of the 2,5-dimethyl-4-hydroxy-3(2H)-furanon, 2-propyltetrahydropyran-3-ol, estragole, benzene, 1-ethynyl-4-fluoro-, 5-hydroxymethylfurfural, anethole, 7oxabicyclo[4.1.0]heptan-2-one,6-methyl-3-(1-methylethyl)-, 2-methoxy-4-vinylphenol, 1,2,2trimethylcyclopentane-1,3-dicarboxylic acid, E-9-tetradecenoic acid, caryophyllene, cholestan-3-ol,2methylene-, (3ß,5α)-, Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-, ß-curcumene, 7-epi-cissesquisabinene hydrate, Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene-,[S-(R*,S*)]-m, octahydrobenzo[b]pyran, 4a-acetoxy-5,5,8a,-trimethyl, dodecanoic acid, 3-hydroxy, tetraacetyl-d-xylonic nitrile, 1-ethenyl 3, trans(1,1-dimethylethyl)-4,cis-methoxycyclohexan-1-ol, phen-1,4-diol,2,3-dimethyl-5trifluoromethyl, 5-benzofuranacetic acid, 6-ethenyl-2,4,5,6,7,7a-hexahydro-3,6-dime, 5-benzofuranacetic acid, 6-ethenyl-2,4,5,6,7,7a-hexahydro-3,6-dime, phytol, acetate, desulphosiniqrin, oxiraneundecanoic acid, 3-pentyl-,methyl ester, cis,Phytol, 9,12,15-Octadecatrienoic acid, 2-phenyl-1,3-dioxan-5-yl ester, butanoic acid, 1a,2,5,5a,6,9,10,10a-octahydro-5,5adihydroxy-4-(h), 9-Octadecenoic acid, 1,2,3propanetriyl ester, (E,E,E) and Diisooctyl phthalate. C. angustifolia was highly active against Aspergillus terreus (6.01±0.27).


INTRODUCTION
Medicinal plants are those plants which contain substances that can be used for the therapeutic purposes in one or more of its organ or substances which are precursors for the synthesis of useful drugs (Sofowora, 1982;Bako et al., 2005;Altameme et al., 2015a).The use of medicinal herbs to relieve and treat diseases is *Corresponding author.E-mail: imad_dna@yahoo.com.Tel: 009647716150716.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License increasing because of their mild features and few side effects (Basgel and Erdemoglu, 2006).These plants are unlicensed and freely available, however, and there is no requirement to demonstrate efficacy, safety or quality (Ernst, 1998).The genus Cassia comprises 580 species of shrubs and trees which are widely distributed throughout the world, of which only twenty species are indigenous to India which belongs to the family Caesalpiniaceae, which generally consist of trees, shrubs and a few woody herbs.Cassia angustifolia Vahl (Family: Caesalpinaceae), popularly known as senna, is a valuable plant drug in Ayurvedic and modern system of medicine for the treatment of constipation.The pods and leaves of senna, as well as the pharmaceutical preparations containing sennosides A and B, are widely used in medicine because of their laxative properties.Senna is used in medicine as a cathartic; it is especially useful in habitual constipation.The laxative property of senna is based on two glycosides viz.sennoside A and sennoside B, whereas sennoside C and D have also been reported in the plant.Apart from sennoside, the pod and leaf also contain glycosides of anthraquinones rhein and chrysophenic acid, recently two naphthalene glycosides have also been isolated from leaves and pods (Gupta, 2010).
Antimicrobial activity has been reported in many plants by various workers (Sarin, 2005;Bansal et al., 2010;Chahal et al., 2010;Seth and Sarin, 2010;Malwal and Sarin, 2011;Hameed et al., 2015a).A new anthraquinone glycoside (emodin 8-0-sophorside) and seven known glycosides were isolated from the leaves of C. angustifolia and their structures were elucidated by spectral analysis (Kinjo et al., 1994).It has antiinflammatory properties (Vanderperren et al., 2005), detoxification ability (Bournemouth, 1992) and also helps improve the function of the digestive system (Hoffmann, 1990).Cassia senna helps to reduce the nervous tension (Mills, 1993) and also helps in aiding the spleen and liver in production of blood and red blood cells (Spiller et al., 2003;Altameme et al., 2015b;Hamza et al., 2015).The present study was undertaken to investigate the antimicrobial activity and phytochemical analysis of C. angustifolia.

Collection and preparation of plant material
C. angustifolia was purchased from local market in Hilla city, middle of Iraq.After thorough cleaning and removal of foreign materials, the seeds were stored in airtight container to avoid the effect of humidity and then stored at room temperature until further use (Hameed et al., 2015b;Jasim et al., 2015).

Preparation of sample
About fifteen grams of methanolic leaves extract of C. angustifolia powdered was soaked in 30 ml methanol for ten hours in a rotatory shaker.Whatman No.1 filter paper was used to separate the extract of plant.The filtrates were used for further phytochemical analysis.It was again filtered through sodium sulphate in order to remove the traces of moisture (Hussein et al., 2015;Hameed et al., 2015c).

Gas chromatographymass spectrum analysis
The GC-MS analysis of the plant extract was made in a Agilent 7890 A instrument under computer control at 70 eV.About 1 μl of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min.As the compounds were separated, they eluted from the column and entered a detector which was capable of creating an electronic signal whenever a compound was detected (Imad et al., 2014a;Kareem et al., 2015).The greater the concentration in the sample, the bigger was the signal obtained which was then processed by a computer.The time from when the injection was made (Initial time) to when elution occurred is referred to as the retention time (RT).While the instrument was run, the computer generated a graph from the signal called chromatogram.Each of the peaks in the chromatogram represented the signal created when a compound eluted from the gas chromatography column into the detector (Mohammed and Imad, 2013;Imad et al., 2014b).The X-axis showed the RT and the Y-axis measured the intensity of the signal to quantify the component in the sample injected.As individual compounds eluted from the gas chromatographic column, they entered the electron ionization (mass spectroscopy) detector, where they were bombarded with a stream of electrons causing, them to break apart into fragments.The fragments obtained were actually charged ions with a certain mass (Hameed et al., 2015d).The mass/charge (M/Z) ratio obtained was calibrated from the graph obtained, which was called the mass spectrum graph which is the fingerprint of a molecule.Before analyzing the extract using gas chromatography and mass spectroscopy, the temperature of the oven, the flow rate of the gas used and the electron gun were programmed initially.The temperature of the oven was maintained at 100°C.Helium gas was used as a carrier as well as an eluent.The flow rate of helium was set to 1 ml per minute.The electron gun of mass detector liberated electrons having energy of about 70eV.The column employed here for the separation of components was Elite 1 (100% dimethyl poly siloxane).The identity of the components in the extracts was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures (Imad et al., 2014c).Compounds were identified by comparing their spectra to those of the Wiley and NIST/EPA/NIH mass spectral libraries.

Determination of antifungal activity
Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 μl of the samples solutions (C.angustifolia) was delivered into the wells.Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.Methanol was used as solvent control.Amphotericin B and fluconazole were used as reference antifungal agent (Hameed et al., 2015b).The tests were carried out in triplicate.The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation.

Statistical analysis
Data were analyzed using analysis of variance (ANOVA), and differences among the means were determined for significance at P

Figure 4 .
Figure 4. Structure of Estragole present in Cassia angustifolia with RT= 6.303 using GC-MS analysis.

Figure 7 .
Figure 7. Structure of Anethole present in Cassia angustifolia with RT= 7.510 using GC-MS analysis.

Figure 12 .
Figure 12.Structure of Caryophyllene present in Cassia angustifolia with RT= 9.301 using GC-MS analysis.

Figure 25 .
Figure 25.Structure of Phytol, acetate present in Cassia angustifolia with RT= 13.953 using GC-MS analysis.

Figure 26 .
Figure 26.Structure of Desulphosiniqrin present in Cassia angustifolia with RT= 14.399 using GC-MS analysis.

Figure 28 .
Figure 28.Structure of Phytol present in Cassia angustifolia with RT= 16.665 using GC-MS analysis.

Figure 32 .
Figure 32.Structure of Diisooctyl phthalate present in Cassia angustifolia with RT= 20.373 using GC-MS analysis.

Figure 34 .
Figure 34.Structure of Squalene present in Cassia angustifolia with RT= 22.604 using GC-MS analysis.

Figure 41 .
Figure 41.Structure of y-Tocopherol present in Cassia angustifolia with RT= 25.236 using GC-MS analysis.

Figure 43 .
Figure 43.Structure of Vitamin E present in Cassia angustifolia with RT= 26.581 using GC-MS analysis.

Figure 44 .
Figure 44.Structure of Campesterol present in Cassia angustifolia with RT= 28.315 using GC-MS analysis.

Table 1 .
Major phytochemical compounds identified in Cassia angustifolia.

Table 2 .
Zone of inhibition (mm) of Aspergillus Spp.test to Cassia angustifolia bioactive compounds and standard antibiotics.