|Year : 2018 | Volume
| Issue : 1 | Page : 15-20
Phytochemical constituents and antimicrobial activity of the ethanol and chloroform crude leaf extracts of Spathiphyllum cannifolium (Dryand. ex Sims) Schott
Dhayalan, Daniel E Gracilla, Renato A Dela Peña Jr, Marilyn T Malison, Christian R Pangilinan
Department of Biology, College of Arts and Sciences, Manila Central University, EDSA, Caloocan City, Philippines
|Date of Web Publication||20-Mar-2018|
Prof. Christian R Pangilinan
Christian Ronquillo Pangilinan, Department of Biology, College of Arts and Sciences, Manila Central University, EDSA, Caloocan City,
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: The study investigated the medicinal properties of Spathiphyllum cannifolium (Dryand. ex Sims) Schott as a possible source of antimicrobial compounds. Materials and Methods: The phytochemical constituents were screened using qualitative methods and the antibacterial and antifungal activities were determined using agar well diffusion method. Statistical Analysis: One-way analysis of variance and Fisher’s least significant difference test were used. Results: The phytochemical screening showed the presence of sterols, flavonoids, alkaloids, saponins, glycosides, and tannins in both ethanol and chloroform leaf extracts, but triterpenes were detected only in the ethanol leaf extract. The antimicrobial assay revealed that the chloroform leaf extract inhibited Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, whereas the ethanol leaf extract inhibited E. coli, S. aureus, and B. subtilis only. The ethanol and chloroform leaf extracts exhibited the highest zone of inhibition against B. subtilis. The antifungal assay showed that both the leaf extracts have no bioactivity against Aspergillus niger and C. albicans. Conclusions: Results suggest that chloroform is the better solvent for the extraction of antimicrobial compounds against the test organisms used in this study. Findings of this research will add new knowledge in advancing drug discovery and development in the Philippines.
Keywords: Antimicrobial, chloroform extract, ethanol extract, phytochemical, Spathiphyllum cannifolium
|How to cite this article:|
, Gracilla DE, Dela Peña Jr RA, Malison MT, Pangilinan CR. Phytochemical constituents and antimicrobial activity of the ethanol and chloroform crude leaf extracts of Spathiphyllum cannifolium (Dryand. ex Sims) Schott. J Pharm Bioall Sci 2018;10:15-20
|How to cite this URL:|
, Gracilla DE, Dela Peña Jr RA, Malison MT, Pangilinan CR. Phytochemical constituents and antimicrobial activity of the ethanol and chloroform crude leaf extracts of Spathiphyllum cannifolium (Dryand. ex Sims) Schott. J Pharm Bioall Sci [serial online] 2018 [cited 2018 Aug 19];10:15-20. Available from: http://www.jpbsonline.org/text.asp?2018/10/1/15/227682
| Introduction|| |
Effective treatment is needed for the global challenge of antibiotic resistance. Nowadays, the major problem doctors are experiencing in providing treatment to patients is the continuous emergence of new strains of bacteria that are resistant to narrow- and broad-spectrum antibiotics resulting in prolonged illness or even death., Antibiotic resistance in bacteria can cause diseases that are even more severe than the nonresistant strains. This development challenges the scientific community to discover new, safe, and more effective antibiotic compounds from natural sources apart from the existing synthetic antibiotic agents because almost all available antibiotics cause side effects and are also expensive. Thus, these considerations make it essential to discover new and more potent antibiotics to address the problem of new and emerging antibiotic-resistant pathogens.
From the beginning of human civilization, nature has been a fundamental source of remedy to many ailments. Traditional medicines of both plant and microbial origin provide safe remedies against diseases as advocated by the World Health Organization. Natural products are helpful in drug development as most clinical drugs originated from natural products including plant secondary metabolites. Although the main role of these secondary metabolites is defense against plant predators and pathogens, interestingly, there are now a huge number of reports that explore the activity of these natural products present in leaves and many other plant parts for pharmacological applications including the development of antimicrobial drugs.,
Spathiphyllum cannifolium is an evergreen, tropical flowering plant widely distributed across Southeast Asia and is commonly cultivated as an ornamental. Previous studies confirmed the plant has anti-inflammatory effect and antibacterial activity against Escherichia More Details coli and Bacillus subtilis., The plant, however, has not been extensively studied in terms of the antimicrobial potentials of compounds from its leaves against a variety of other pathogens, including fungi. Hence, the present study intends to investigate the phytoconstituents and the antibacterial and antifungal potentials of the secondary metabolites produced in the leaves of the plant of interest. Results of the study may provide bases for further investigations involving antibiotic-resistant strains of the test organisms.
| Materials and Methods|| |
Collection and identification of plant materials
Plant leaf materials weighing about 2kg were collected from the grounds of Manila Central University, Caloocan City, the Philippines, and subjected to solvent extraction. A plant leaf, flower, and photographs of the specimen were sent to the Botany Division of the Museum of Natural History, Manila, the Philippines, for taxonomic identification.
Preparation of ethanol and chloroform leaf extracts
The plant materials were washed thoroughly with tap water followed by sterilized distilled water for the removal of dust and dirt. The leaves were shade dried for 28 days at room temperature and then finely powdered using an electric grinder. The finely powdered leaves were divided into two equal parts. These were soaked in ethanol and chloroform, respectively, and kept in a dark room for 72h. Then, the mixtures were filtered using Whatman filter paper (Sigma-Aldrich, St. Louis, MO) no. 2 and the filtrates were subjected to rotary evaporation at 45°C to eliminate the solvents used for extraction.
Qualitative analysis was performed to detect the presence of plant secondary metabolites in S. cannifolium as described by Evans and Guevarra, with minor modifications.
Liebermann–Burchard Test for sterols and triterpenes: Two grams of the concentrated extract was dissolved with acetic anhydride, and the soluble portion was decanted. Two drops of concentrated sulfuric acid were added into the decanted solution and observed for color change. Blue indicates the presence of sterols, whereas red indicates the presence of triterpenes. In the Salkowski’s test for sterol, concentrated sulfuric acid was added into the test substance taken with 2mL of chloroform, and two drops of acetic anhydride were added. Positive reaction is indicated by red coloration in the chloroform layer.
Mg + turning test for flavonoids: Dried sample weighing about 10g was defatted with petroleum ether until the extract is almost colorless. Then, the petroleum ether was discarded. Subsequently, small amount of the defatted sample was treated with 1.0mL of 10% HCl and magnesium turnings. Positive result is indicated by red coloration.
Mayer’s test for alkaloids: Two grams of the concentrated extract was treated with 1% HCl and then filtered. Two drops of Mayer’s reagent were added into the filtrate and a cream precipitate was observed as an indication of a positive result. Results were verified using Wagner’s test by dissolving a small amount of the sample with 1.0mL dilute acetic acid. A white or cream precipitate indicates positive reaction.
Froth test for saponins: Two grams of the concentrated extract was dissolved with hot water and mixed. The mixture was filtered and the filtrate was agitated vigorously and a frothing that lasted for at least 30min indicated a positive result. This was confirmed using hemolysis of red blood cell (RBC) test by dissolving 2g concentrated extract with 80% ethanol; this was mixed and filtered. Subsequently, the filtrate was poured into standard red blood cell suspension. Positive reaction is indicated by hemolysis in which the red color of the RBCs disappeared.
Fehling’s test for glycosides: Two grams of the concentrated extract was dissolved with hot water and mixed. The mixture was then filtered, and the filtrate was divided into two parts and placed into separate test tubes. Dilute HCl (1mL) was added into the first test tube, whereas the second tube served as the control. Afterward, the test tubes were placed in a boiling water bath for 5min and allowed to cool before neutralization. Then, sufficient amount of anhydrous sodium carbonate was added until no effervescence is produced. After neutralization, 1mL of Fehling’s solution was added to both tubes and then reheated in a water bath for 2min. A brick-red precipitate indicates positive reaction. The amount of precipitate in the first test tube was compared with that of the second tube.
Ferric chloride test for tannins: The concentrated extract (2g) was dissolved with hot water and mixed. The mixture was filtered and equal portion of the filtrate was added into two test tubes. To the first portion of the extract was added two drops of ferric chloride. Production of dark precipitate that can be either dark green or blue green indicates positive result.
Evaluation of the antimicrobial activity
Source of microorganisms: The test bacteria, E. coli and Staphylococcus aureus, were clinical isolates previously identified using biochemical analyses. Strains of B. subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 27853 were obtained from the Department of Microbiology, University of the Philippines, Manila, and the test fungi, Aspergillus niger UPCC 4219 and Candida albicans UPCC 2168, were provided by the Natural Science Research Institute, University of the Philippines, Diliman, Quezon City.
Antibacterial test: The agar well diffusion method was used to assess in vitro antibacterial activity of the plant extracts. Approximately 15mL of sterile Mueller–Hinton agar (HiMedia, Mumbai, India) was poured into sterile disposable Petri dish More Detailses and inoculated with bacterial culture test suspension previously prepared by inoculating sterile 0.1% peptone water (HiMedia, Mumbai, India) with each bacterium and then compared to a McFarland standard to give a 1.5×108 cfu/mL bacterial concentration. Then, wells with a diameter of 10mm were punched using a sterile cork borer. Subsequently, the wells were impregnated with 100 µL of the crude extracts and controls. Tetracycline solution (1mg/mL) was used as a positive control. The plates were allowed to stand for 30min to ensure proper diffusion of the extracts before incubation at 37°C for 24h. After incubation, the zone of inhibition indicated by a halo around the wells was measured using a Vernier scale in millimeters., The procedure was conducted at the Biotechnology Laboratory of Manila Central University, Caloocan City.
Antifungal test: The antifungal assessment of the plant extracts was performed following the agar well diffusion method. The culture media used was glucose yeast peptone agar for the yeast C. albicans and potato dextrose agar for the mold A. niger. The wells in this part were impregnated with 200 µL of the plant extracts and controls. Clotrimazole (Canesten [Bayer, Berkshire]) solution (1mg/mL) was used as positive control for both C. albicans and A. niger. The C. albicans plates were incubated at 30°C for 24h, whereas the A. niger plates were incubated at 25°C for 3 days. The antifungal procedure was carried out at the Natural Science Research Institute of the University of the Philippines, Diliman, Quezon City.
Statistical analysis: The results obtained were subjected to one-way analysis of variance (ANOVA) using GraphPad Prism (GraphPad Software, La Jolla, CA) version 6. The comparison among the means was carried out using Fisher’s least significant difference (LSD) test.
| Results|| |
Qualitative analysis [Table 1] of the plant secondary metabolites in S. cannifolium ethanol leaf extract revealed the presence of sterols in trace amount. Alkaloids and glycosides were detected to be moderately abundant, whereas triterpenes, flavonoids, saponins, and tannins were abundant. In the chloroform extract, saponins were detected in trace amount; flavonoids, alkaloids, and glycosides were found to be moderately abundant; and the presence of sterols was found to be abundant. Triterpenes were not detected in the chloroform extract. This suggests that more polar secondary metabolites than nonpolar were extracted with the solvents used in this study.
|Table 1: Qualitative analysis of the phytochemical constituents of S. cannifolium|
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Inhibitory activity of the leaf extracts of S. cannifolium against C. albicans UPCC 2168 and A. niger UPCC 4219
[Figure 1] shows the mean inhibition zones produced by the extracts against fungal test organisms. The chloroform leaf extract (T2) of S. cannifolium and clotrimazole (Canesten), the positive control (T+), inhibited C. albicans with a mean diameter of 14.67mm and 32.0mm, respectively. ANOVA revealed that the zone of inhibition produced by the test extracts and control groups in C. albicans has significant difference at 95% confidence level with the P value <0.0001. The dimethyl sulfoxide (DMSO) solvent control, distilled water (T−), and ethanol extract (T1) did not inhibit C. albicans. Multiple comparison using Fisher’s LSD test revealed that T+ is still significantly higher than T2. The inhibitory effect exhibited by T2 against C. albicans suggests that it is capable of inhibiting yeast species. Also, both the ethanol and the chloroform leaf extracts of S. cannifolium did not inhibit A. niger. The results suggest that S. cannifolium synthesizes nonpolar compounds that have inhibitory activity against yeasts but not against mold species.
|Figure 1: Mean diameter of zones of inhibition produced by the extracts and controls against the fungal test organisms|
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Inhibitory activity of the leaf extracts of S. cannifolium against selected bacterial pathogens
[Figure 2] shows the mean inhibition zones produced by the leaf extracts against all test bacteria. The data revealed that both the ethanol and the chloroform leaf extracts of S. cannifolium (T1 and T2) and the positive control tetracycline (T+) exhibited varying zones of inhibition against all bacteria tested, whereas no inhibition was observed in DMSO, chloroform solvent controls, and in T− (distilled water). The mean inhibition produced by the ethanol leaf extract, chloroform leaf extract, and positive control against S. aureus was 16.0, 14.67, and 35.33mm, respectively. ANOVA revealed that the zone of inhibition produced by the test extracts and control groups in all bacteria tested has significant difference at 95% confidence level with the P value of <0.0001. Multiple comparison using LSD shows that the ethanol and the chloroform leaf extracts were statistically comparable. This is indicated by the same letter in the figure and that both the test extracts were significantly higher than the negative control. However, the positive control is still significantly higher than the test extracts. Similarly, the test extracts showed antibacterial activity against E. coli with a mean zone of inhibition of 16.30mm and 18.30mm for the ethanol and the chloroform leaf extracts, respectively. Also, the positive control produced 31.70-mm inhibition zone. Multiple comparison using LSD shows that the test extracts were significantly higher than the negative control and that both extracts exhibited statistically similar effect. However, the positive control has a statistically higher zone of inhibition. The mean zone of inhibition produced by the ethanol and the chloroform leaf extracts against B. subtilis was 24.33mm and 16.67mm, respectively, whereas the positive control produced 28.33mm. Multiple comparison revealed that the ethanol leaf extract has significantly higher antibacterial activity against B. subtilis than the chloroform extract. Both extracts were significantly higher than the negative control but shows significantly lower activity than the positive control. Between the two extracts tested, only the chloroform extract showed inhibitory activity against P. aeruginosa with a mean diameter of 15.67mm, whereas the positive control produced 27.17mm. Multiple comparison using LSD showed that the chloroform extract has significantly lower antibacterial activity compared to the positive control.
|Figure 2: Mean number of zone of inhibitions produced by the extracts and controls against (A) S. aureus, (B) E. coli, (C) B. subtilis ATCC 6633, and (D) P. aeruginosa ATCC 27853|
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| Discussion|| |
A variety of secondary plant metabolites were detected in the leaves of S. cannifolium extracted using two solvents, namely, ethanol (polar) and chloroform (nonpolar). These include sterols, flavonoids, alkaloids, saponins, glycosides, and tannins that were all detected in both extracts, whereas triterpene was detected only in ethanol extract. The presence of these phytoconstituents supports the significant bioactivity exhibited by the crude extracts against the microorganisms tested. According to Compean and Ynalvez and Wink, plant secondary metabolites are associated with many bioactivities including inhibitory properties against a wide range of pathogens. Among these secondary metabolites, alkaloids have been studied extensively in terms of their antimicrobial activities and mechanism of action. Similarly, sterols, flavonoids, tannins, saponins, glycosides, and triterpenes have been reported to have significant inhibitory properties against different pathogens.
Our results differed from a previous study that reported methanol and ethyl acetate crude leaf extracts of S. cannifolium exhibited antibacterial activity against B. subtilis but not against E. coli.,
| Conclusions|| |
The leaves of S. cannifolium are rich in phytoconstituents as shown in the qualitative analysis. The presence of sterols, flavonoids, alkaloids, saponins, glycosides, and tannins in both ethanol and chloroform leaf extracts was detected, whereas triterpenes were only present in the ethanol leaf extract. Results of the antimicrobial analyses revealed that the chloroform leaf extract exerted significant bioactivity against E. coli, S. aureus, B. subtilis, and P. aeruginosa, whereas the ethanol leaf extract was bioactive against E. coli, S. aureus, and B. subtilis only. The ethanol and chloroform leaf extracts exhibited the highest zone of inhibition against B. subtilis. Finally, although the chloroform extract was bioactive against C. albicans, both leaf extracts showed negative results against A. niger. The phytochemicals present in the ethanol and chloroform crude extracts could account for the bioactivity observed against the test organisms. Further studies involving purification and isolation of the phytoconstituents in the leaf extracts and antimicrobial testing against multidrug-resistant strains are needed for further drug discovery.
Presentation at a meeting: Biology Teacher’s Association of the Philippines in Philippine Normal University, Philippines on April 6, 2017.
Financial support and sponsorship
Conflict of interest
The authors declare no conflict of interest.
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[Figure 1], [Figure 2]