WSN 157 (2021) 25-37 EISSN 2392-2192
Evaluation of phytochemical composition and
in vitro antioxidant potential of Cyathea latebrosa
leaves
Chinyere B. C. Ikpa1, Tochukwu D. O. Maduka1,*, Chinomnso Uzoamaka Ikpa2
1Department of Pure and Industrial Chemistry, Imo State University, PMB 2000, Owerri, Imo Atate, Nigeria
2Department of Biochemistry, Alex Ekwueme Federal University, Ndufu-Alike Ikwo, P.M.B. 1010, Abakaliki, Ebonyi State, Nigeria
*E-mail address: davemaduluv@gmail.com
ABSTRACT
Phytochemicals and antioxidant potentials of chloroform extract of leaves of Cyathea latebrosa have been investigated. The metabolites detected are Carbohydrates, starch, glycosides, saponins, alkaloids, tannins, flavonoids, sterols, terpenes, and polyuronides. Quantitative Phytochemical analysis gave alkaloid (0.08×102 ±0.02), saponins (0.16×102 ±0.02), tannin (0.11×102 ±0.01), flavonoid (0.16×102 ±0.01), and phenol (0.18×102 ±0.01). FT-IR (KBr) cm-1 analysis showed -NH-(3433.41 weak), -OH- (3851.01 sharp), -C-F (1408.8 strong) –SO-(1101.39) and Ar (1646.3 weak), GC/MS characterization revealed the extract contained [2-Aziridinylethyl]amine (70.7%), Benzeneethamine, 3- fluoro-β,5-dihydroxy-N-methyl (14.0%), N-Methyltaurine (10.7%) and (R)-(-)-2-Amino-1-propanol (3.15%). The presence of these compounds exposed the leaves as a potential remedy for alleviated cold, cancer, malaria and to regulate immune functions. At 400 µg/ml concentration the extract and ascorbic acid produced 54.34% and 96.02% antioxidant activity respectively. The FRAP values were significantly (p < 0.05) low than the value of ascorbic acid (2µ M). The plant extracts showed low antioxidant potential even on higher concentrations when compared to reference standard ascorbic acid.
Keywords: Phytochemical, Characterization, GC/MS analysis, Treatment, antioxidant, Cyathea latebrosa, phytochemical composition, in vitro
1. INTRODUCTION
Medicinal plants have been used by all mankind as a source of medicines since ancient times. In recent times, there have been growing interests in exploring the biological activities of different medicinal herbs due to their natural origin, cost-effectiveness, and lesser side effects1. Interest in medicinal plants as a re-emerging health aid in the maintenance of personal health and well-being has been fuelled by rising costs of prescription drugs and the bioprospecting of new plant-derived drugs2. Generally, plants that produce constituents having medicinal values are called medicinal plants. These substances differ from plant to plant, thus the plant kingdom provides a large store of various chemical substances with potential therapeutic properties which have been utilized in the treatment and cure of human and other animal diseases including relieving pains, convulsion, and cardiovascular diseases3 among others. Drugs of natural origin are considered to be less toxic and free from adverse effects than synthetic ones. Even though the active compounds of many herbal drugs are unknown, they have been widely prescribed by the practitioners of the traditional medicines due to their minimal adverse effects and low cost4.
The ethnomedicinal studies revealed that ferns can be used for the treatment of diseases of rheumatisms, lungs, blood, digestion, and gynaecology5. A disease causal of utmost concern is the free radicals that are delivered as a consequence of typical biochemical responses in the body. The free radicals are involved in cancer generation, ischemic heart disease, inflammation, diabetes, aging, atherosclerosis, immunosuppression, and neurodegenerative disorders 6, 7. Literature on the use of the secondary metabolites of the plant e.g flavonoids and terpenoids as an important agents in the defense against free radicals has been widely reported 8, 9. The medicinal plant in this study is a fern; while the bioactivities of ferns have been previously investigated, little attention has been given to the Family of Cyatheaceae 9. Although, some species in the genera of Cyatheaceae have been employed in traditional remedies for various diseases; at present, literature on the phytochemical profile of Cyathea latebrosa has been understudied worldwide.
Cythea latebrosa also called tree fern is a primitive fern in the order Cyatheales, characterized by ascending trunk-like stems and arborescent habit, these ferns have a medium growth rate with a height of 8-10m, and meanwhile, they are conspicuous plants of humid tropical region 10. Chai et al in 2013 had reported on the antioxidant activity of cyathea latebrosa when they investigated the antibacterial, anti-glucosidase, and antioxidant activities of four (4) highland ferns of Malaysia. HowYee Lai and YauYan Lim also reported strong antioxidant properties in Cyathea latebrosa in 2011, when they screened the methanolic extracts of fifteen fern species, both investigations were done in South East Asia, Malaysia. The lack of literature on the medicinal capabilities of these ferns on African soils has prompted this timely research. Hence, we attempted to fill the gaps in current knowledge about the therapeutic potentials of Cyathea latebrosa by adding further investigations.
We have investigated the functional groups present in the extract of this species alongside the phytochemical and antioxidant investigations. Consequently, this work has been designed to explore the phytochemicals present in the fern Cyathea latebrosa; carrying out both qualitative and quantitative tests and spectral analysis using the Infra-red spectra and gas chromatography-mass spectrometric methods. The antioxidant properties of the extracts have been investigated through analysis of the ferric reducing antioxidant power (Frap) and radical scavenging activity (dpph) assays.
2. METHODOLOGY
2. 1. Identification and preparation of plant leaves
Matured leaves of Cyathea latebrosa, identified by Prof. Onu, M.O. of IMSU Owerri Nigeria was harvested from a palm plantation at Ehime Mbano L.G.A. Imo State, Nigeria. The leaves sample was dried at room temperature and ground with a new (unused) mortar and pestle.
The powdered leaves 300g were soaked with 1.5 liters of ethanol and filtered. The crude ethanolic fraction was partitioned with water and chloroform and the organic fraction (chloroform) was further analyzed using GC/MS and FT-IR to obtain the chemical composition.
2. 2. Phytochemical Analysis 2. 2. 1. Qualitative Phytochemistry
The plant extract was assessed for the existence of the phytochemicals using the method of Treas & Evans 11. Both primary and secondary Metabolites were investigated.
2. 2. 2. Quantitative phytochemistry
Determination of alkaloids: This was done by the gravimetric method. Powdered leaves of C. latebrosa 5g were soaked in 50 ml ethanol containing 10% acetic acid. The mixture was filtered after 4h and concentrated by evaporating 75% of the solvent. Conc. NH4OH was added drop-wise to precipitate the alkaloids. The alkaloid was filtered, washed, weighed and the percentage determined.
Determination of tannins: Powdered leave sample 0.5g was extracted with 10 ml of H2O and added to 35ml of H2O in a 50 ml conical flask. 5 ml of tannic acid was treated in the same manner. The mixture was allowed to stand for 1.5h and the absorbance was taken at 760 nm to obtain a concentration of tannin.
Determination of Phenols: 0.2g of sample was soaked with 10 ml methanol and filtered.
1ml of the filtrate was mixed 1 ml of Folin Ciecalteau reagent and 2 ml of 20% Na2CO3 solution was added. A standard phenol was treated in the same way and the color absorbance was recorded at 560 nm.
Determination of saponins: The powdered leave sample 10g was soaked in 100 ml of 20%
ethanol. The suspension was heated in a hot water bath for 4h with continuous mechanical stirring at 55 ºC. The combined extract was reduced to 20 ml over a water bath at 90ºC. The concentrate was transferred into a separating funnel and 10ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was washed twice with 5 ml of 5% aqueous NaCl. The remaining solution was heated in the water bath to evaporate it. After evaporation, the residue was weighed dried in an oven to a constant weight and the percentage saponin was determined.
Determination of flavonoids: The powdered sample 20 mg was extracted with 50 ml of 80% aqueous methanol at room temperature. The solution was transferred into a crucible and evaporated to dryness over a water bath and weighed percentage flavonoids was calculated.
2. 2. 3. IR and GC/MS Analysis
IR spectra were recorded on KBr (cm-1) with SHIMAZU FT-IR 8400s FOURIER TRANSFORMER INFRARED SPECTROPHOTOMETER NARIT Zaria. GC/MS analysis was recorded with Agillant 7890AGCMS of Halden Company Port Harcourt, Rivers State.
2. 2. 4. In vitro free radical scavenging assays
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Photometric Assay
The free radical scavenging activity of the extract was analyzed by DPPH Assay simulating the procedure used by Mensor et al 12 with slight modifications. The crude extract at concentrations (25, 50, 100, 200, and 400) µg/ml each was mixed with 1 ml of 0.5 m M DPPH (in methanol) in a cuvette. The absorbance at 517 nm was taken after 30 minutes of incubation in the dark at room temperature. The analysis was done in triplicate. The percentage antioxidant activities were calculated as follows.
% antioxidant activity (AA) = 100 − [{(ABS sample—ABS blank)×100}
ABS control ]
1.0 ml of methanol plus 2.0 ml of the test extract was used as the blank while 1.0 ml of the 0.5mM DPPH solution plus 2.0 ml of methanol was used as the negative control. Ascorbic acid was used as the reference standard (Iwalewa et al) 13.
2. 2. 5. Ferric Reducing Antioxidant Power
The ferric-reducing antioxidant power was carried out as described by Benzie and Strain
[14]. The FRAP working solution was freshly prepared in each test. The aqueous solution of a known amount of ascorbic acid was used for calibration. Colorimetric readings were recorded at 593 nm, at 37 ºC. The ascorbic acid standard solution was tested in a parallel process.
Calculations were made by a calibration curve. The value of the ferric reducing antioxidant power was calculated as follows
FRAP value of sample (µMol/L) =
changes in absorbance of sample from 4 min − 0mins FRAP value of std (1000μm) Changes in absorbance of STD 4 min − 0 min
2. 2. 6. Data analysis
Statistical analysis was done in triplicates and data was reported as mean ± standard errors. The data analysis was analysed using the one-way ANOVA test and the Tukey HSD test was used to identify any significance between the values of the samples at the 0.05 level of probability.
3. RESULTS
3. 1. The phytochemical screening
Results from the investigation on the extract of Cyathea latebrosa for different classes of phytochemicals were presented in Tables 1 and 2, Qualitative phytochemical screening was carried out by setting up a set of qualitative characterization reactions. The results of this phytochemical screening are reported in Table 1. The qualitative Phytochemical screening tests have revealed the presence of Carbohydrates, starch, Glycosides, Saponins, Alkaloids, Tannins, Flavonoids, alkaloids, Terpenes, and Polyuronides. The quantitative phytochemical screening revealed high concentrations of flavonoids, phenols, and tannins.
Table 1. Results of the qualitative phytochemical analysis of C. latebrosa.
Phytochemical Result
Carbohydrate +
Starch +
Glycosides +
saponins +
Alkaloids +
Tannins +
flavonoids +
alkaloids +
Terpenes +
Polyuronides +
Table 2. Results of the quantitative phytochemical analysis of C. latebrosa
Phytochemicals % Concentration Alkaloid 0.08×102 ±0.02 Flavonoid 0.16×102 ±0.01 Saponin 0.02×102 ±0.02
Phenol 0.18×102 ±0.01
Tannin 0.11×102 ±0.01
The phytochemical results showed the presence of alkaloids, flavonoids, saponin, phenol, and tannins at different concentrations.
3. 2. FT-IR analysis
FT-IR spectrum of the chloroform fraction of C. latebrosa is displayed and presented in Fig. 1 and Table 3; the result of the analysis revealed the presence of the following functional groups; -SO-, R-F, Ar, and –OH. The peaks that are stretched and pointed are said to be strong while the dwarf peaks are referred to as weak peaks.
Figure 1. FTIR spectrum of C. latebrosa leaves
Table 3. Results FT-IR Analysis of C. latebrosa leaves
S/N Functional groups Peaks Comments
1 -SO- 1101.3
2 R-F 1408.08 Strong
3 Ar 1646.3 Weak
4 -NH- 3433.41 Weak
5 -OH- 3851.01
3. 3. GC-MS analysis
The GC-MS chromatogram and mass spectrum of chloroform extract of C. latebrosa are presented in Table 2. GC-MS analysis resulted in the identification of 4 chemical compounds.
The retention time, molecular formula, and the % content are presented in Table 4. GC/MS characterization shows that the extract contained [2-Aziridinylethyl] amine (70.7%), Benzeneethamine, 3-fluoro-β,5-dihydroxy-N-methyl (14.0%), N-Methyltaurine (10.7%) and (R)-(-)-2-Amino-1-propanol (3.15%).
Table 4. Result of GC/MS Analysis of C. latebrosa leaves
Compds Name M.
weight
M.
formula
Base peak
R.
index % content R. Time/min 1 [2-Aziridinylethyl] amine 86 C4H10N2 45 14533 70.7 30.75 min
2
Benzeneethanamine-3- fluro-β-5-dehydroxy-N-
methyl
185 C9H12FNO 42 14308 14.0 30.755 min
3 N-methyl-taurine 139 C3H9NO3S 64 14722 10.7 26.936 min
4 2-amino-I-propanol 75 C3H9NO 42 3386 3.15 30.755 min
M. weight = molecular weight, M. formula = molecular formula, R. index = Retention index, R. Time/min = Retention Time/min
3. 4. The antioxidant activity of Cyathea latebrosa
The result of the in vitro antioxidant activity using DPPH scavenging activity and ferric reducing antioxidant power (FRAP) of the chloroform extract of Cyathea latebrosa was presented in Table 5; Fig. 2 and 3 shows the graphical representation of the result. The extract produced a significant (p < 0.05) concentration-dependent increase in percentage antioxidant
activity in the DPPH photometric assay. At 400 µg/ml concentration the extract and ascorbic acid produced 54.34% and 96.02% antioxidant activity respectively. The antioxidant activity of the extract was significantly (p <0.05) lower than the antioxidant activity of the ascorbic activity. In the FRAP result, the extract at 25, 50, 100, 200, and 400 µg/ ml concentration reduced 0.25, 0.38, 0.48, 0.99 and 1.66 µM of Fe3+ to Fe2+ respectively.
Table 5. DPPH scavenging activity and ferric reducing antioxidant power (FRAP) of the extract of Cyathea latebrosa.
Concentration (µg/ml )
DPPH antioxidant activity C. latebrosa Ascorbic acid
Frap
25 6.83 ± 3.44 72.58 ±5.20 0.24 ± 0.04 50 10.39 ± 0.96 97.20 ± 0.33 0.37 ± 0.01 100 20.05 ± 3.00 95.58 ± 0.07 0.47± 0.03 200 29.66 ± 0.54 95.61 ± 0.22 0.99 ± 0.03 400 54.34 ± 1.85 96.07 ± 0.06 1.65 ± 0.11
Figure 2. The in vitro antioxidant effect of Cyathea latebrosa using frap assay.
0 0,5 1 1,5 2 2,5
25 50 100 200 400 125
FRAP value in µM ±SEM
Concentration µg/ml Cyathea latebrosa
Ascorbic acid
Figure 3. The in vitro antioxidant effect of Cyathea latebrosa on dpph assay
4. DISCUSSION
The result of the phytochemical screening (Table 1 and 2) agrees with the report of earlier literature on some ferns 15-17. The tables showed the presence of flavonoid which are potent water-soluble, antioxidants and free radical scavengers, which prevents cell damage and have anti-cancer properties18. Tannin properties contribute to the use of plant leaves in the treatment of ulcers and wounds. Alkaloids and phenols support the traditional use of the plant to treat bacteria and fungi infections 19.
The indications of –OH-, -NH-, R-F, -SO- functional groups on FT-IR result (Table 2) support the medicinal properties of the plant leaves. The GC/MS analysis (Table 3) gave the presence of the following four compounds which have some pharmaceutical properties.
Compound 1:
N H2
N
[2-Aziridinylethyl]amine
[2-Aziridinylethyl]amine is an amine compound that has been used to synthesis many pharmaceutical compounds. [2-Aziridinylethyl]amine derivatives have been applied in the treatment and prevention of many diseases20.
0 20 40 60 80 100 120
25 50 100 200 400
Percentage antioxidant activity ±SeM
Concentration µg/ml
Cyathea latebrosa Ascorbic acid
Compound 2:
NH O
H
F
OH
Benzeneethamine, 3-fluoro-β,5-dihydroxy-N-methyl is applied medicinally in the treatment of cold, pains, and weight loss.
Compound 3:
O
S OH O
N H
N-Methyltuarine
N-Methyltaurine showed chemo preventives against colon and hepatic cancer, used as anti- bacteria, anti-malaria and regulates immune functions21.
Compound 4:
(R)-(-)-2-Amino-1-propanol is used to boost immunity and also for water treatment22.
DPPH assay usually measures the electron-donating potential of the sample to the stable DPPH radical 23. A concentration-dependent increase in antioxidant activity was observed in the result and this is similar to a study by Chai et al. 9. Although, the antioxidant activity of the extract was significantly (p <0.05) lower than the antioxidant activity of the ascorbic acid. The inhibition concentration of 50% IC50 of the extract corroborated with the result of a similar
investigation made by other researchers on the same plant 9, 24-34. Monitoring the ability of an antioxidant to donate an electron to Fe(III) resulting in the reduction of Fe3+ complex to Fe2+
complex has also been a method to detect antioxidant potential. The higher the FRAP value, the greater the reducing power of the tested compound, thus the greater the antioxidant activity.
The FRAP values of the extract produced a significant (p < 0.05) concentration-dependent increase and were significantly (p < 0.05) lower when compared with the value of ascorbic acid control (2µ M). This points out that although the plant possesses antioxidant potentials, it cannot replace already discovered antioxidants. The result in the study also corroborates with the result reported by chai et al 25.
5. CONCLUSION
This study supports the usefulness of Cyathea latebrosa leaf and its medicinal properties.
This partly explains the use of this plant material in herbal medicine for the treatment and prevention of the symptoms of some diseases.
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