|Year : 2020 | Volume
| Issue : 4 | Page : 368-374
Comparative analysis of the antimicrobial potential of stem and fruit extracts of Calotropis procera
Sharafa Adegoke Bello1, Tawakalt Adedayo Ayofe1, Mohammed Faridat Yakub1, Abdullahi Temitope Jamiu2
1 Department of Biological Sciences, Al-Hikmah University, Ilorin, Kwara, Nigeria
2 Department of Biological Sciences, Al-Hikmah University, Ilorin, Kwara, Nigeria; Department of Microbial Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa
|Date of Submission||10-Jun-2020|
|Date of Acceptance||24-Aug-2020|
|Date of Web Publication||23-Jan-2021|
Mr. Abdullahi Temitope Jamiu
Department of Biological Sciences, Al-Hikmah University, P.M.B. 1601, Ilorin, Kwara; Department of Microbial Biochemical and Food Biotechnology, University of the Free State, Bloemfontein
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The dramatic rise in antimicrobial resistance continues to threaten the effective management and treatment of emerging and re-emerging infectious diseases. Medicinal plants offer excellent therapeutic alternative especially due to their plethora bioactive constituents and low resistance development to them. Purpose: The comparative analysis of the antimicrobial potential of the stem and fruit extracts of Calotropis procera was investigated. Materials and Methods: Using different extracts of the plant, the phytochemical screening was determined alongside their antimicrobial properties with agar-disc diffusion assay. The antimicrobial potency of the plant extracts (200 mg/ml) was evaluated and compared by their inhibition zone (IZ), activity index (AI), percentage specific activity (PSA), and percentage total activity (PTA) values. Results: The highest antibacterial activity (IZ = 15 ± 0.5 mm) was displayed by the stem hot aqueous extract against Staphylococcus aureus, while the maximum antifungal effect was exerted by both the stem cold and hot aqueous extracts (P > 0.05). The overall antimicrobial AI (AI = 1.56) was displayed by the stem cold aqueous extract against Candida albicans. The stem's hot and ethanolic extracts exhibited the highest achievable PSA of 100'. Furthermore, the stem extracts displayed the PTA of 83.3' compared to 25' by the fruit extracts, thus confirming the greater antimicrobial potency of the plant's stem extracts. Conclusion: This study suggests that while the stem extracts of C. procera could have displayed better antimicrobial activity, the overall effects elicited by the plant could be attributed to the presence of phytochemicals as revealed by the result of the phytochemical screening. Further studies focusing on complete characterization and evaluation of the mechanism of antimicrobial action of bioactive constituents of the extracts is underway.
Keywords: Antimicrobial activity, antimicrobial resistance, Calotropis procera, medicinal plants, phytochemicals
|How to cite this article:|
Bello SA, Ayofe TA, Yakub MF, Jamiu AT. Comparative analysis of the antimicrobial potential of stem and fruit extracts of Calotropis procera. Phcog Res 2020;12:368-74
|How to cite this URL:|
Bello SA, Ayofe TA, Yakub MF, Jamiu AT. Comparative analysis of the antimicrobial potential of stem and fruit extracts of Calotropis procera. Phcog Res [serial online] 2020 [cited 2021 Mar 9];12:368-74. Available from: http://www.phcogres.com/text.asp?2020/12/4/368/307651
| Summary|| |
- The antimicrobial activity of the stem and fruit extracts of Calotropis procera against Staphylococcus aureus, Escherichia More Details coli, and Candida albicans was compared. The plant extracts contain phytochemicals such as saponins, tannins, flavonoids, alkaloids, phenols, steroids, phytosterols, and terpenoids. The antimicrobial potential of the stem extracts was found to be greater that of the fruit extract, thus suggesting the better efficacy of the stem extracts. Further studies focusing on complete characterization and evaluation of the mechanism of antimicrobial action of bioactive constituents of the extracts is underway.
Abbreviations Used: CSC: Calotropis procera stem cold aqueous extract; CSH: Calotropis procera stem hot aqueous extract; CSE: Calotropis procera stem ethanolic extract; CSM: Calotropis procera stem methanolic extract; CFC: Calotropis procera fruit cold aqueous extract; CFH: Calotropis procera fruit hot aqueous extract; CFE: Calotropis procera fruit ethanolic extract; CFM: Calotropis procera fruit methanolic extract; IZ: inhibition zone; AI: activity index; PSA: percentage specific activity; PTA: Percentage total activity.
| Introduction|| |
The landmark discovery of the first medical miracle, penicillin, by Sir Alexander Fleming in 1928 marked the modern era of antibiotics., Since then, antibiotics have been used to improve the quality of lives, save lives, and reduce economic burden posed by infectious diseases., However, the inappropriate use and overuse of antibiotics in human and veterinary medicine have led to dramatically increased resistance in both bacteria and fungi. Antimicrobial resistance (AMR) is an eminent public health crisis responsible for reduced quality of lives, increased hospitalization, and increased economic burden. The impacts of AMR in developing nations like Nigeria are even more amplified due to inadequate medical resources., Furthermore, it has been estimated that, by 2050, some 10 million annual mortality will be attributable to AMR, if no practical alternatives are sought. In pursuit of novel and alternative treatment options, natural sources such as medicinal plants are being explored and could serve as possible sources of novel and affordable antimicrobial agents.,
Plants have been harnessed as a medicinal source since primordial times. The traditional and folk medicines utilize plant products for the treatment of various infectious diseases. In addition, plants have also been shown to possess anticancer, anti-inflammatory, immunomodulatory, antimicrobial, and antioxidant properties.,, These properties elicited by plants are due to their plethora of bioactive constituents such as alkaloids, flavonoids, tannins, saponins, terpenoids, sugars, amino acids, proteins, et cetera., Furthermore, several studies have lent credence to the potential use of plant materials for novel drug discovery and development.,,
Calotropis procera (Ait.) R. Br., with common names, Sodom apple, swallowwort, dead sea apple, milkweed (English), kisher (Arabic), pomme de sodome (French), bomubomu (Yoruba), and tumfafiya (Hausa), is a wild-growing plant that belongs to the family Asclepiadaceae. It is endemic to and widely distributed in the tropical and subtropical regions of Africa and Asia., Traditionally, C. procera is used to treat ailments such as fever, rheumatism, pain, asthma, bronchitis, ulcer, indigestion, cold, eczema, measles, diarrhea, abscesses, and jaundice., More specifically, its stem is used for the treatment of elephantiasis, cough, asthma, dysentery, and skin diseases (e.g., eczema and leprosy). The leaf possesses analgesic and antinociceptive properties and is also used in snakebite antidote. [Table 1] highlights few pharmacological properties of the plant.
Although studies have reported the antimicrobial properties of various parts of C. procera, none provided a comprehensive comparison of the antimicrobial property of the stem and fruit extracts of the plant.,,,, The present study attempted to provide a comprehensive report on the comparative analysis of the antimicrobial potential of the stem and fruit extracts of C. procera.
| Materials and Methods|| |
Already identified and characterized clinical isolates, including Staphylococcus aureus, Escherichia coli, and Candida albicans, were obtained from the Microbiology Laboratory, Al-Hikmah University, Ilorin. The isolates were maintained on agar slants at 4°C for future use.
Collection and preparation of plant materials
Fresh and healthy stems and fruits of C. procera [Figure 1] were collected from a garden near Al-Hikmah University (8.482” N, 4.505” E), Ilorin, Nigeria. The plant materials were washed, air-dried, and pulverized into a fine homogeneous powder using a sterile ceramic mortar and pestle. The powdered material was weighed and kept in an air-tight container before extraction.
|Figure 1: Calotropis procera plant showing its long and slender stem (a) and fruit (b)|
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Preparation of plant extracts
The plant extraction was done as earlier described with slight modifications. Briefly, 70 g powdered plant materials were prepared separately in 700 ml of low-to-high polarity solvents, namely ethanol, methanol, and distilled water (cold and hot); the plant extraction was done using a rotary shaker for 48 h. The resulting extract was filtered with a sterile filter paper (Whatman No. 1), and the filtrate was evaporated and dried using a water bath at 40°C for 48 h. The concentrated extract was subsequently scraped into a sterile bottle with a sterile spatula and stored at 4°C for future use.
Qualitative phytochemical screening
The phytochemical (saponins, tannins, flavonoids, alkaloids, phenols, steroids, and phytosterols) constituents of all different plant extracts which include C. procera stem cold aqueous extract (CSC), C. procera stem hot aqueous extract (CSH), C. procera stem ethanolic extract (CSE), C. procera stem methanolic extract (CSM), C. procera fruit cold aqueous extract (CFC), C. procera fruit hot aqueous extract (CFH), C. procera fruit ethanolic extract (CFE), and C. procera fruit methanolic extract (CFM) were qualitatively determined as described below.
Test for saponins
This was done by frothing test with few modifications of previous method. Briefly, 50 mg of each plant extract was dissolved in distilled water (5 ml) and filtered. The resulting filtrate was homogenized vigorously and warmed (10 min). The formation of a stable foam confirmed the presence of saponins.
Detection of tannins
Briefly, 50 mg of each extract was mixed with distilled water (2 ml). This was filtered and accompanied by the addition of few drops of 10' FeCl3 solution to the filtrate. A brownish-green or blue-black coloration/precipitation when compared with the control indicates the presence of tannins.,
Detection of flavonoids
Briefly, 25 mg of each extract was dissolved in 2 ml diluted NaOH. This was filtered and followed by the addition of few drops of hydrochloric acid (HCl) into the filtrate. A yellow coloration when compared with the control compared indicated the presence of flavonoids.
Detection of alkaloids
Briefly, 25 mg of each extract was dissolved with 3 ml of 1' HCl; the mixture was heated and then filtered. Mayer's and Wagner's reagents were added to the resulting filtrate. A yellowish precipitate confirmed the presence of alkaloids.
Detection of phenols
Briefly, 50 mg of each extract was dissolved in distilled water (2 ml), filtered, and accompanied by the addition of ferric chloride solution (2 ml) to the filtrate. The formation of blue-black or brown color confirmed the presence of phenols.
Detection of phytosteroids
Briefly, 50 mg of each extract was dissolved in 2 ml chloroform and filtered, and two drops of concentrated sulfuric acid were added to the filtrate. This was homogenized and left for some minutes. The presence of a golden yellow color when compared with the control confirmed the presence of phytosteroids.
Detection of steroids
For this, Liebermann–Burchard's test was performed. Briefly, 50 mg of each extract was dissolved in 5 ml chloroform and filtered; 2 ml sulfuric acid and 2 ml acetic anhydride were serially added to the resulting filtrate. A color change from violet to blue-green when compared with the control sample confirmed the presence of steroids.,
Test for terpenoids
Briefly, 50 mg of each extract was dissolved in 2 ml chloroform and filtered, and 2 ml sulfuric acid was subsequently added to solution. Formation of reddish-violet color when compared with the control was an indication of terpenoids' presence.
Standard antibiotic and antifungal susceptibility assay
This was done to determine the susceptibility profiles of the tested isolates and was carried out using the agar-disc diffusion method. Briefly, overnight nutrient broth cultures of each bacterial strain were standardized to a turbidity of 0.5 McFarland standard, and standardized yeast culture (OD600 = 0.5) was also prepared. Dried surface of Mueller-Hinton agar (MHA) or Sabouraud's dextrose agar (SDA) plate was swabbed with standardized test isolate's inoculum (100 μl). Commercially purchased antibiotics discs or antifungal-loaded discs were placed onto the agar surface, and the plates were incubated at 37°C (24 h) and 37°C (24–48 h) for the bacterial and fungal strains, respectively. The inhibition zone (IZ) was measured in millimeter and was compared with CLSI antibiogram standards.,,
Evaluation of the antimicrobial activity of plant extracts
The antimicrobial activity of the C. procera stem and fruit's extracts was evaluated using the agar-disc diffusion method. Briefly, 200 mg/ml concentration of each extract was prepared by dissolving 0.4 g of respective extract in distilled water (2 ml). Subsequently, overnight nutrient broth cultures of each bacterial strain were standardized to a turbidity of 0.5 McFarland standard. The standardized culture (100 μl) of each bacterial strain was evenly spread on MHA surface using a sterile hockey stick. Standard discs (6?mm in diameter) impregnated with plant extract (200 mg/ml) were equidistantly placed onto the agar and incubated at 37°C for 24 h. For the yeast strain, 100 μl of an overnight Sabouraud's dextrose broth (SDB) culture (OD600 = 0.5) of C. albicans was evenly spread on SDA surface. Subsequently, the plant extract-loaded discs (200 mg/ml) were placed onto the agar and incubated at 37°C for 24–48 h. Discs of gentamicin (10 μg/ml) and ketoconazole (25 μg/ml) were used as standard drugs (positive controls) for the antibacterial and antifungal assays, respectively. Negative controls were set up with discs impregnated with sterile distilled water. The antimicrobial activity was recorded as the diameter of IZ formed around the discs.,,
Other antimicrobial activity indexes
The activity index (AI) of each plant extract relative to the drug standard was calculated to express the relationship between the extract and the reference drug.,
The percentage specific activity (PSA) to determine the antimicrobial activity of each extract against all tested isolates was calculated.
While the percentage total activity (PTA) of the extracts of each plant material (i.e., stem or fruit) was also evaluated, this was important to determine the overall antimicrobial activity of each plant material. As an example, the PTA for the stem extracts was calculated as:
The data obtained with respect to the zones of inhibition (mean ± standard deviation) of the extracts were compared with that of standard drugs using one-way analysis of variance complemented with Tukey's multiple comparisons test (www.graphpad.com). A significant difference was taken at P < 0.05 and indicated on the graph by different letters.
| Results|| |
Qualitative phytochemical screening
The results of phytochemical screening of C. procera stem and fruit extracts are presented in [Table 2]. The presence of saponins, tannins, flavonoids, alkaloids, phenols, steroids, phytosterols, and terpenoids was observed in at least one of the plant extracts. However, tannins and steroids were absent in all the stem extracts, while the fruit extracts were all devoid of alkaloids [Table 2]. Moreover, saponins, phenols, and flavonoids appear to be the most abundant secondary metabolites in the plant materials evident by their presence in at least six out of the eight extracts screened.
Standard antibiotic and antifungal susceptibility assay
The antimicrobial susceptibility profiles of the tested isolates against standard antibiotics and antifungal drugs are shown in [Table 3]. The S. aureus strain used in this study was resistant to six out of the eight standard antibiotics tested, thus indicating its multidrug resistance phenotype. However, the E. coli strain used was only resistant to ceftazidime and augmentin. In addition, the yeast strain used (C. albicans) in this study was susceptible to nystatin and ketoconazole but was resistant to the most commonly used antifungal drug, fluconazole.
|Table 3: Antimicrobial susceptibility profiles of tested isolates with standard drugs|
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Evaluation of the antimicrobial activity of plant extracts
The antimicrobial potential of the plant extracts was determined using the agar-disc diffusion method. The three microbial strains tested were sensitive to at least one of the plant extracts, excluding the CFC and CFE. The highest activity (IZ = 15 ± 0.5 mm) against S. aureus was recorded for CSH, and this compared favorably with that exerted by gentamicin (P > 0.05) [Figure 2]a. However, CSC, CFC, CFH, CFE, and CFM had no antibacterial effects against S. aureus (P > 0.05). For E. coli, CSC exhibited the highest antimicrobial activity (IZ = 9 ± 0.50 mm) among all extracts screened [Figure 2]b. This was followed by CSH, CSE, and CFH (P > 0.05), however, the standard drug (gentamicin) had the overall highest activity (IZ = 17 ± 0.0 mm) against E. coli (P < 0.05) [Figure 2]b. Moreover, the tested E. coli strain was markedly resistant to CSM, CFC, CFE, and CFM (P > 0.05).
|Figure 2: Antibacterial activity of Calotropis procera stem and fruit extracts against (a) Staphylococcus aureus and (b) Escherichia coli bars with different letters being significantly different (P < 0.05)|
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As shown in [Figure 3], among all the eight extracts and one standard drug (ketoconazole) screened against the yeast strain, CSC and CSH displayed the most pronounced antifungal activities (IZ = 25 ± 0.5 mm and IZ = 23 ± 2.0 mm, respectively) (P > 0.05). The antifungal effect (IZ = 16 ± 0.5 mm) of the standard drug was favorably comparable to that exerted by CSE, CSM, and CFM (P > 0.05) but slightly higher than what was displayed by CFH (IZ = 10 ± 0.5 mm) (P < 0.05). However, the yeast strain was resistant to CFC and CFE (P > 0.05).
|Figure 3: Antifungal effect of Calotropis procera stem and fruit extracts on Candida albicans|
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Antimicrobial susceptibility indexes such as AI, PSA, and PTA were used to compare the antimicrobial effects of the stem and fruit extracts of C. procera against the tested isolates [Table 4]. The highest antibacterial AI was recorded for CSH (AI = 0.88) against S. aureus. This was followed by AI of 0.59 and 0.41 for CSE and CSM, respectively, against the same organism. The maximum antifungal AI against the yeast was exhibited by CSC (AI = 1.56), followed by CSH (AI = 1.44) and CSE (AI = 1.06).
|Table 4: Antimicrobial activities of Calotropis procera stem and fruit extracts against all tested microbial strains|
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The highest PSA of 100' was recorded for CSH and CSE, indicating that the two extracts were active against all tested isolates. This was followed by PSA of 66.7' for CSC, CSM, and CFH, respectively. However, no activity was displayed by CFC and CFE against all the tested isolates. Finally, the comparison of the PTA of the stem and fruit extracts, as shown in Table 4, indicates that the stem extracts exhibited the overall highest PTA of 83.3', while that of the fruit extracts was 25.0', thus indicating the better activity of the stem extracts.
| Discussion|| |
The dramatic rise in AMR development continues to threaten the effective management and treatment of emerging and re-emerging infectious diseases, and this consequently increases the morbidity, mortality, and economic burdens associated with such infections. Medicinal plants represent an alternative, inexpensive, and rich source of antimicrobial agents due to the profusion of bioactive compounds they contain., In the present study, the antimicrobial activity of the stem and fruit extracts of C. procera against S. aureus, E. coli, and C. albicans was assayed, while the antimicrobial potentials of the two plant parts were also compared. Aside from the dramatic rise and increased threat displayed by AMR, the clinical isolates used during this study were selected because of their clinical significance; S. aureus is the leading etiology of both nosocomial and community-acquired infections and largely implicated in septicemia, pneumonia, and wound infections; E. coli is a prominent cause of enteritis and urinary tract infection, while C. albicans remains the predominant cause of invasive fungal infections.
The qualitative phytochemical screening of C. procera stem and fruit extracts revealed the presence of saponins, tannins, flavonoids, alkaloids, phenols, steroids, phytosterols, and terpenoids in at least one of the plant extracts [Table 2]. The presence of these secondary metabolites in the plant extracts may explain their antimicrobial effects since secondary metabolites are known to possess antimicrobial, anticancer, antimalarial, and antioxidant properties.,,, Furthermore, the presence of saponins in the methanolic extracts of the fruit and stem of C. procera agrees with previous findings., On the other hand, the absence of alkaloids in the methanolic extracts of both fruit and stem also coheres with findings of previous study. Moreover, the presence of flavonoids in the aqueous extracts of the plant stem is consistent with the report of a previous study.
The antimicrobial susceptibility patterns of the tested isolates with standard drugs revealed that the S. aureus used in this study is probably a multidrug-resistant (MDR) strain due to its resistance to six out of the eight tested antibiotics. The MDR phenotype displayed by this strain may be due to the synthesis of β-lactamase which causes hydrolysis of β-lactam ring, thus resulting in inactivation of β-lactam antibiotics. On the other hand, the E. coli strain was only resistant to two out of the eight tested antibiotics. Moreover, the yeast strain was resistant to fluconazole, which is the most commonly used antifungal drug due to its low toxicity, great efficacy, high bioavailability, and high-water solubility.
In general, all the plant extracts screened displayed enormous antimicrobial property, with only two out of the eight extracts screened exhibiting no antibacterial or antifungal property. The highest antibacterial activity (IZ = 15 ± 0.5 mm) was displayed by CSH against S. aureus, while the maximum antifungal effect was exerted by CSC and CSH (P > 0.05).
The antimicrobial potential of the plant extracts was also assessed by evaluating AI of each extract relative to standard drug. Gentamicin was used as the reference drug for the bacterial isolates, while ketoconazole was used for the yeast strain. For this, the best antibacterial AI was exhibited by CSH (AI = 0.88) against S. aureus, while the maximum antifungal (AI = 1.56) against the yeast was displayed by CSH. This indicates that the overall antimicrobial (AI = 1.56) was displayed by CSH. The highest achievable PSA of 100' was exhibited by CSH and CSE; this suggests the broad-spectrum activities of these two extracts against all tested isolates. However, the CFC and CFE had a specific activity of 0' which translates to their non-antimicrobial effects against all the tested strains. Furthermore, the stem extracts displayed the PTA of 83.3' compared to 25' exhibited by the fruit extracts, thus suggesting the better efficacy and greater antimicrobial potency of the plant's stem extracts.
| Conclusion|| |
Our present study reveals and confirms the plethora of phytochemical constituents of C. procera stem and fruit's extracts. It also suggests the better potency and efficacy of the stem extracts compared with the fruit extracts of the plant. These findings validate and expand our current knowledge of the antimicrobial potentials of medicinal plants, plant products, and secondary metabolites. However, further studies should focus on complete characterization and evaluation of the mechanism of antimicrobial action of bioactive constituents of the extracts.
The authors acknowledge with thanks the laboratory technologists and assistants at Microbiology Laboratory, Al-Hikmah University, for the assistance rendered during laboratory analysis. The authors also thank Dr. Sabiu Saheed of the Durban University of Technology, South Africa, for his technical advice and support.
Financial support and sponsorship
None relevant to this study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]