Brazilian Journal of Biological Sciences, 2016, v. 3, no. 5, p. 209-219. ISSN 2358-2731 http://dx.doi.org/10.21472/bjbs.030519
Mass spectroscopic and phytochemical screening of phenolic compounds in the leaf extract of Senna alata (L.) Roxb. (Fabales: Fabaceae) Solomon Oluwole Oladeji¹,*, Funmilayo Enitan Adelowo¹ and Kehinde Abraham Odelade²
¹Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, P.M.B 4000, Ogbomoso, Nigeria. Email: [email protected]. ²Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P.M.B 4000, Ogbomoso, Nigeria.
Abstract. Senna alata (L.) Roxb. (Fabales: Fabaceae) is a medicinal plant basically used as antifungal and sometimes as antibacterial. Received Local people believe it is the amount of the plant consumed that May 31, 2016 constitutes to its potency, other believe it is the incantation thereby disregarding the bioactive components present in the leaf of S. alata. Accepted June 22, 2016 Therefore, there is a need to examine this claim by examining the bioactive components that are present in the plant. The methanolic Released and ethanolic extracts were obtained using soxhlet apparatus and the June 30, 2016 concentrated extracts were purified using column chromatography; the fractions were eluted and screened for their phytochemical and Open Acess Full Text Article the mass spectroscopic analysis was performed using a mass spectrophotometer. The antimicrobial activity was carried out using agar disc diffusion method. The phytochemical analysis revealed the presence of important secondary metabolites such as anthraquinone, flavonoid and saponins while steroids was absent in the leaf extracts. The molecular ions of 250, 250, and 222 were obtained from the mass spectra. This showed the presence of methaqualone, cinnamic acid and isoquinoline. Ethanolic extracts showed a higher antimicrobial activity when compared with the methanolic extracts but less activity when compared with the standard used (amoxicillin). It could be concluded that the presence of these phytochemicals could be responsible for the observed antifungal and ORCID 0000-0003-3355-2488 antibacterial activities on the susceptible organisms studied of the Solomon Oluwole plant and also can be a natural source of antimicrobial substances of Oladeji high importance. 0000-0002-1949-2418 Funmilayo Enitan Adelowo Keywords: Therapeutics; Herbal medicine; Metabolite; 0000-0003-3380-7997 Spectroscopy. Kehinde Abraham Odelade
Introduction bush. Wild senna (S. alata) is found in Ghana and Brazil, but it is now widely Senna alata (L.) Roxb. is a distributed in the United States of America medicinal plant of Fabaceae family. It has and all over Africa, including Nigeria many common names such as Candle bush, (Farnsworth and Bunyapraphatsara, 1992). Acapulo, Ringworm bush and Calabra
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The plant is very important in many Spectroscopy, nuclear magnetic resonance areas of life. The applications are so spectroscopy (NMR), Liquid numerous. The applications include for Chromatography-Mass Spectrometry (LC- medicinal purposes, antimicrobial activities, MS), mass spectrometry (MS), and X-ray antioxidant activities, its nutritional values crystallography. If the compound of interest and many others. Different parts and is already known, it can be identified with constituents of the plant are reported to less measurement by comparing its exhibit several therapeutic properties, such characteristic features with literature values as antibacterial, antifungal, antimicrobial or the data of standard compounds. and analgesic (Igoli et al., 2005; Makinde et The quantification of active al., 2007), antioxidants (Juvekar and compounds in medicinal plants have Halade, 2006). The biological activity of become very significant, it has been shown plants is basically due to the presence of that in-vitro screening methods could certain structural feature of a compound or provide the needed preliminary its metabolite. One of these features is the observations necessary to select crude plant effect of structural features like conjugated extracts with potentially useful properties double bonds; this dictates UV absorption for further chemical and pharmacological properties of some flavonoids or phenolic investigations (De Fatima et al., 2006). The compounds. Alternatively, a certain activity phenolic compound in S. alata varied from may depend on the stereochemistry of the the leaf to the flower and this led to compound since target enzymes and different approaches by the scientists to biological systems in general are stereo- screen the plant for its therapeutic potency specific. Therefore, it is inevitable to (Owoyale et al., 2005). In recent research characterize the compounds present in plant carried out on S. alata plant, it was materials. discovered that it contain hundreds of Phenolic compounds are extracted bioactive compounds (De Fatima et al., and purified or clean up from the plant 2006). The compositions of these material before structural characterization compounds are not the same and also, it of the compounds. First, the metabolic was discovered that different bioactive activity of the plant is halted by flash compounds were present and this dictate its freezing and lyophilization or by extraction therapeutic functions. of the plant materials using solvent of high Plants produce a large number of polarity such as methanol, ethanol and chemical substances or metabolites. In acetone. The samples were pulverized and attempt to investigate these secondary subjected to pre-treatment before extraction metabolites, the extraction was done using in order to improve the extraction yield. soxhlet apparatus and also, identification The crude extract may be purified using and structural elucidation and the cleanup methods such as adsorption, antimicrobial screening of the partition, gel permeation, ion exchange etc phytochemical present in the leaf of S. alata and also, column chromatographic methods were investigated. such as gel filtration over Sephadex LH-20 and reversed-phase (RP) chromatography Materials and methods (Waterman and Mole, 1994; Wang et al., 1998). There are many physicochemical Solvents methods used for the identification of a The solvents used for extraction specific compound. This is usually and clean up (ethanol and n-hexane) were achieved by a combination of several purified by distillation and the fraction physicochemical methods, such as collected at their boiling point. ultraviolet spectroscopy (UV), gas chromatography-mass spectrometry (GC- Collection of sample MS), circular-dichroism spectroscopy (CD), The leaves of S. alata were Thin-Layer Chromatography, optical collected in a farm in Odokoto Area, rotation, Fourier Transform Infrared (FTIR) Ogbomoso, Nigeria, between May and
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Mass spectroscopic and phytochemical screening in the leaf extract of Senna alata 211
August, 2015, and identified by the Antimicrobial screening herbarium unit of the Department of Pure Antibacterial screening. The and Applied Biology, LAUTECH, antibacterial assay was carried out using Ogbomoso. The leaves samples of S. alata nutrient agar (NA) as the media. Each NA were rinsed with distilled water and spread plate contains different isolate. About 6 mm on a flat surface to air-dried for 25 days. diameter filter paper discs were sterilized at 160 °C for 2 h. Methanol and ethanol are Sample preparation used as control for methanolic and The dried leaves samples were ethanolic extracts. The zone of inhibition of pulverized and sieved using a sieve of mesh the bacterial spores was measured after size of 20 mm. The sieved samples were incubating at 37 oC for 24 h. The minimum stored in air-tight containers and stored at concentration of each extract that inhibits 40C for further analysis. the growth of the bacterium was taken as the Minimum Inhibitory Concentration Extraction and concentration (M. I. C.). Soxhlet apparatus was used for the extraction and was carried out based on the Antifungal screening. The method of Adelowo and Oladeji (2016) antifungal assay was carried out using with little modification. The leaf extract potato dextrose agar (PDA) as the media. was concentrated using rotary evaporator. Each PDA plate contains different isolate. About 6 mm diameter filter paper discs Clean up were sterilized in Petri dishes at 160 oC for The column used was made of 2 h. Methanol and ethanol are used as Pyrex glass, and have small diameter so as control for methanolic and ethanolic to have effective separation and obtain extracts. The zone of inhibition of the distinctive bands. The cleanup method was fungal spores was measured after carried out based on the method of incubating at 30 oC for 3 days. The Adelowo and Oladeji (2016). Ethanol and minimum concentration of each extract that n-hexane (2:1 vol: 30 mL) were used as the inhibits the growth of the fungus was taken eluting solvent. The fractions were eluted as the Minimum Inhibitory Concentration and were collected in different beakers for (M. I. C.). subsequent analyses. Results and discussion Phytochemical screening of phenolic compound The phytochemicals properties of The phytochemical analysis of the S. alata leaf extracts were carried out for the The phytochemical analysis presence of bio-molecular compounds such indicated the presence of important as anthraquinone, flavonoids, tannins, secondary metabolites (tannins, saponins, alkaloids and steroids using the standard flavonoids, anthraquinone). The presence of qualitative procedure as described by bioactive components screened showed that Owoyale et al. (2005). S. alata plant posses antimicrobial properties. The result of phytochemical Mass spectroscopic analysis of analysis showed that S. alata contained phenolic compounds tannins, steroids, phenols and saponins The MS analysis of the fractions (Table 1). Medicinal plants contain a wide obtained from S. alata plant extracts was variety of secondary metabolites or performed according to the method of compounds such as tannins terpernoids, Adelowo and Oladeji (2016). alkaloids, flavonoids; that dictates the
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Table 1. The phytochemicals properties of methanolic and ethanolic S. alata leaf (+ (present) and - (absent)). Phytochemicals Methanolic extract Ethanolic extract Tannins + + Saponins + + Anthraquinone + + Steroids + _ Flavonoids + +
therapeutic potency of the plants most detergents, pesticides and molluscides in especially the antimicrobial activities (Sule addition to their industrial applications such et al., 2010). as foaming and surface active agents. They The therapeutic potentials of S. help in controlling cardiovascular diseases alata plant can be linked to the presence of and in controlling cholesterol in humans these bioactive components. Similar Onwuliri et al. (2004). In addition to their phytochemical constituents such as use in industry, saponins also have a wide flavonoids and tannins were also revealed range of medicinal applications (Sule et al., to be active against pathogenic bacteria 2010). such as Bacillus cereus, Staphylococcus aureus amongst others (Kumar et al., 2012). Mass spectrometric analysis of The tannins present in the leaf of S. alata phenolic compounds in S. alata leaf make it useful in production of antiseptic The mass spectra of the phenolic soap which are commonly used in bathing compounds in S. alata leaf extracts were or cleansing of skin surfaces. Documented determined by conventional electron impact literatures have it that phytochemicals can ionization. In the mass spectrum of each be toxic to filamentous fungi, yeasts and compound, the molecular ion, base peak bacteria (Treese and Evan, 2004), and also, and the fragmentation of the compound inhibitory to viral reverse transcriptase were shown. (Onwuliri et al., 2004). A wide range of physiological activity of saponins, steroids, Mass spectrometric analysis of phenols and tannins are found to be more methaqualone predominant and therefore may be Methaqualone (Figure 4) gave responsible for the antimicrobial action relatively weak molecular ions under (Sule et al., 2010). The leaf of the S. alata electron impact conditions except the base plant also showed the presence of peak which gave a relative density of Flavonoids, they are discovered to be a 100%. The observed fragment ions of group of polyphenolic compounds that methaqualone are shown in Table 2 with showed antifungal, antibacterial, anticancer their respective m/z values and their and anticancer potency Ekpo et al. (2010). relative intensity. The possible Tannins have astringent properties fragmentation pattern of methaqualone is which hasten the healing of wounds and given in scheme 1 (Figure 3). The fragment inflamed mucous membrane due to their showed m/z values of 65, 91, 132 and 235. physiological activities such as anti- Also, the mass spectrum of methaqualone is oxidant, antimicrobial and anti- given in Figure 2. In the spectrum of inflammatory properties. The use of S. methaqualone, the m/z value of 235 gave alata leaves directly for healing fungal the highest relative intensity due to the loss . infections has long been in practice. of CH3 from the molecular ion of m/z of Saponins have been traditionally used in 250.
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Mass spectroscopic and phytochemical screening in the leaf extract of Senna alata 213
Figura 3. Mass spectrum of methaqualone.
Figure 4. Fragmentation pattern of methaqualone (Scheme 1).
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Table 2. Principal ions in the electron impact mass spectrum of methaqualone C6H4C=ON2C2H3C6H4R [R=CH3]. Fragment ion(s) m/z value(s) Relative intensity + -CH3H2CH3] 65 15 + -C6H3C=O-] 91 45 + [C6H4C=ON2C2H3C6H4- 235 100 + [C6H4C=ON2- 132 15
. the loss of –Si(CH3)3H2] from the molecular ion was observed in the spectrum, this lead to the m/z value of 175 (Table 3). From the mass spectrum (Figure 7), the m/z value for the cinnamic acid [m/z 250] gave the base peak. The loss
-CH=CH-C6H4-O-Si(CH3)3] corresponding to the m/z 177 from the molecular ion. Also observed was a prominent peak due to the loss of the CH3 radical, which showed a Figure 2. Methaqualone structure. relative intensity of 75%.
Mass spectrometric analysis of cinnamic acid Cinnamic acid (Figure 5) gave a strong molecular ion under Electron Impact condition. Fragment ions were also observed at m/z 235, 203, 175, 115, 89, 73 and 59. The fragmentation pattern of this compound is represented in scheme 2 Figure 5. Cinnamic acid structure. (Figure 6). A weak peak corresponding to
Figure 6. Mass spectrum of cinnamic acid.
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Figure 7. Fragmentation pattern of cinnamic acid (scheme 2).
Table 3. Principal ions in the Electron Impact mass spectrum of Cinnamic acid, p- (trimethylsiloxy)-, methyl ester. Fragment Ion(s) m/z value (s) Relative Intensity (%) + -CH3-O-C=O] 59 20 + -Si(CH3)3] 73 65 + -O-Si(CH3)3] 89 25 + [CH3-O-C=O -CH=CH-C6H2- 175 15 + -C-CH=CH-C6H4-O-Si_(CH3)3] 203 50 + -O-C=O-CH=CH-C6H4-O-Si(CH3)3] 235 75
Mass spectrometric analysis of and 77 corresponding to the loss of some isoquinoline radicals from the molecular ion were Isoquinoline (Figure 10) gives the observed. The fragmentation pattern of principal ions in the mass spectrum of Isoquinoline is given in scheme 3 (Figure Isoquinoline (Figure 8) were given in Table 9). The base peak from the spectrum 4. For the entire fragments observed, the (Figure 9) was obtained by the loss of molecular ion observed was a weak peak. -CH2C6H3-OH-O-CH3] (corresponding to The molecular ion observed showed m/z m/z value of 137) from the molecular ion. value of 222 with a low relative intensity. Also, the m/z 118 was obtained from the
The base peak from the spectrum is loss of –C2H5-NH-CH2-C6H3OH-O-CH3] to observed with m/z value of 162. Prominent give 25% relative intensity. peaks of m/z values of 252, 190, 162, l18
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Figure 8. Mass spectrum of isoquinoline.
Figure 9. Fragmentation pattern of isoquinoline (Scheme 3).
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Table 4. Principal ions observed in the electron impact mass spectrum of isoquinoline. Fragment ion(s) m/z value (s) Relative Intensity (%) + -C2H5-OH-OCH3] 77 15 + [CH3-O-C6H3-C- 118 25 + [CH3-O-C6H3-C3H5-NH- 162 100 + [CH3-O-C6H3-C3H5-NH-CH2-C6H4- 252 0
(Khan et al., 2001; Abubacker et al., 2008). The result of this study revealed the effect of ethanol fractions on Candida albicans to be significantly higher than the methanol leaf which thus similar to report of Ogunti and Olujoba, (1993) where the ethanolic fraction was found to exhibit marked antimicrobial activity against Aspergillus niger and Candida albicans when compared to methanolic fractions. The Figure 10. Isoquinoline structure. extracts showed potency when compared
with the standard antibiotics used Antimicrobial activities of (Amoxicillin). The presence of these phenolic compounds in S. alata leaf phenolic compounds has been found to be extracts responsible for the fungicidal activity The growth of fungi and bacteria is (Khan et al., 2001). Variations in the a measure of fungitoxicity and concentrations of the phenolic compounds bacteriotoxicity of the fungicide and in ethanolic and methanolic fractions are bactericide respectively. The minimum responsible for the differences in the concentration (MIC) obtained from the fungicidal activity between the methanolic activities of the test organisms against the and ethanolic (Timothy et al., 2012). The fraction and amoxicillin are given in Table Minimum Inhibitory Concentration 5. obtained showed that Aspergillus niger and The ethanolic and methanolic of S. Candida albicans are more susceptible to alata plant showed antibacterial and the ethanolic leaf even at low concentration antifungal properties and all tested bacterial when compared with Staphylococcus and fungal isolates responded positively to aureus, Bacillus cereus and Klebsiella spp. the extracts. The minimum inhibitory This study justifies the ethno medical use of concentrations are shown in Table 5.There this plant in the inhibition of bacterial and are tremendous microbial effects on other fungal diseases. We believe that the Aspergillus niger and Candida albican results obtained from this study are an compared to the corresponding bacteria encouragement for further studies that will used. This agrees well with several reports lead to the elucidation of the structure of in which similar observations were made the active components.
Table 5. Inhibitory effect of the fractions on the growth of fungi and bacteria species expressed through MIC in μg/mL. Test organisms/ fractions ME leaf EE leaf Amoxicillin Staphylococcus aureus 19 21 25 Bacillus cereus 16 20 25 Klebsiella spp 18 22 24 Aspergillus niger 24 29 30 Candida albican 25 28 29 Where: ME= Methanolic, EE= Ethanolic (units are in millimeters).
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Conclusion Farnsworth, N. R.; Bunyapraphatsara, N. Thai Medicinal Plant: recommended for Primary In this study, the mass Health Care System. Bangkok, Thailand: spectroscopic and phytochemical analysis Prachachon Company, 1992. of S. alata was investigated. The analyses Igoli, J.; Ogaji, O.; Igoli, N. Tor-Anyiin T. A. showed that the plant contains saponins, Traditional medicinal practices among the Igede reducing sugars, flavonoids, methaqualone, people of Nigeria (part II). African Journal of Traditional Complementary and Alternative cinnamic acid, toluidine, terpenes, Medicine, v. 2, p. 134-152, 2005. isoquinoline, anthraquinone and glycosides Juvekar, R.; Halade, G. V. Hypoglycemic which could be responsible for the activity of Cassia auriculata in neonatal therapeutic potency of the plant. The streptozotocin-induced non-insulin dependent highest concentration of phenolic diabetes mellitus in rats. Journal of Natural compounds in the extracts was obtained Remedy, v. 6, no. 1, p. 14-8, 2006. using solvents of high polarity; the Khan, M.; Kihara, M.; Omoloso, A. methanolic extract manifested greater Antimicrobial activity of Cassia alata. power of extraction for phenolic Fitoterapia, v. 72, p. 561-564, 2001. compounds from S. alata. The results of the Kumar, A.; Shukla, R.; Singh, P.; Prasad, C. S.; study suggested the great value of the Dubey, N. K. Assessment of Thymus vulgaris L. species S. alata for use in pharmacy and essential oil as a safe botanical preservative phytotherapy; therefore, it could serve as a against post harvest fungal infestation of food natural source of antimicrobial substances commodities. Innovation Food Sci. Emerg., of high importance. v. 9, no. 4, p. 575-580, 1984. http://dx.doi.org/10.1016/j.ifset.2007.12.005 Acknowledgements Makinde, A.; Igoli, J.; Amal, L.; Shaibu, S.; Garbal, A. Antimicrobial activity of Cassia The authors want to appreciate the alata. African Journal of Biotechnology, v. 6, efforts of Doctor Olugbenga Bello of the p. 1509-1510, 2007. Department of Pure and Applied Ogunti, E.; Olujoba, A. Laxative activity of Chemistry, LAUTECH, Ogbomoso, Cassia alata. Fitoterapia. Economical Botany, Nigeria, for his contributions during the v. 64, p. 437-439, 1993. course of the study. Onwuliri, F. C. Antimicrobial studies of the extracts of Acalypha wllkesiana L. on microorganisms associated with wound and skin Conflict of Interest infections. West Africa Journal of Biological Science, v. 15, p. 15-19, 2004. Authors declare that they have no Owoyale, J.; Olatunji, G.; Oguntoye, S. conflict of interests. Antifungal and antibacterial activities of an ethanolic extract of Senna alata leaves. Journal References of Applied Science and Environmental Management, v. 9, p. 105-107, 2005. Abubacker, M.; Ramanathan, R.; Senthil, K.T. Owoyale, J. A.; Olatunji, G. A.; Oguntoye, In vitro antifungal activities of Cassia alata S. O. Antifungal and antibacterial activities of Linn. flower extract. , v. Natural Product Rad. an alcoholic extract of Senna alata leaves. 7, p. 6-9, 2008. Journal of Applied Science and Adelowo, F. E.; Oladeji, S. O. Environmental Management, v. 9, no. 3, Spectrophotometric analysis of phenolic p. 105-107, 2005. Available from: compounds in Senna alata. Am. J. Adv. Sci.
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