COMPARATIVE PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY OF CALOTROPIS SP. OF ETHNOMEDICINAL SIGNIFICANCEHTML Full Text
COMPARATIVE PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITY OF CALOTROPIS SP. OF ETHNOMEDICINAL SIGNIFICANCE
Rasmita Sethy and Bandana Kullu *
Department of Botany, Utkal University, Bhubaneswar - 751004, Odisha, India.
ABSTRACT: The global prevalence of infectious diseases caused by microorganisms is a major public health concern worldwide. The emergence of antibiotic resistance and related toxicity issues are limiting the use of antibiotics and prompting research for alternative therapy. Plant-derived drugs are gaining popularity due to their efficacy and safety. Calotropis procera and Calotropis gigantea are two common species in the genus Calotropis, well known for their ethnomedicinal values. Present study comparatively evaluated the phytochemical screening and antimicrobial activity of C. procera and C. gigantea. The qualitative phytochemical screening of the ethanolic, methanolic, and aqueous extracts of Calotropis sp. indicated similar phyto-constituents of both the species. Secondary metabolites such as alkaloids, terpenoids, flavonoids, glycosides, cardiac glycosides, coumarin, steroid, phenol, tannin, saponin, and volatile oils were detected in both the plant extracts and ethanolic extracts contained the highest number of the plant metabolites. The plant extracts showed antimicrobial activity against Gram +ve and –ve human pathogenic bacteria, indicating their broad-spectrum activity. Comparatively, C. procera was more effective against the tested bacterial pathogens with lower MIC and MBC value than C. gigantea. The study suggested that aerial plant parts extracts of Calotropis sp. would provide therapeutic phytochemicals as anti-microbial agents and can serve as a viable alternative source of bioactive compounds with pharmaceutical relevance.
Calotropis procera, Calotropis gigantea, Phytochemical screening, Anti-microbial activity, MIC, MBC
INTRODUCTION: Infectious diseases are the cause of mortality and morbidity, accounting for more than about 22% of the global disease burden 1. Discovery of antibiotics, the wonder drug in the 20th century, is one among the significant achievements in medical science against infectious diseases.
However, the indiscriminate use of antibiotics has led to the development of antibiotic-resistant bacteria population at alarming frequency 2, 3. Additionally, antibiotics are also known to cause adverse side effects on the host 4. In the current scenario, antibiotic resistance has increased substantially and is developing as an ever-increasing therapeutic challenge. Thus, alternative therapy is the need of the time. Plants are the basis of traditional medicine which have been in use since ancient time and are continuing to provide new remedies to mankind 5. Hence, screening of safe and potent natural antimicrobial agents from plants is increasing throughout the world 6, 7.
Recently for the development of alternative therapy workers emphasized the control of antibiotic-resistant bacterial pathogens using phytochemicals 8, 9.
Calotropis a genus of flowering plants in the family Asclepiadaceae, is one among the native plants of India. Geographically it is widely distributed in southern Asia, northern Africa and northeastern South America. Calotropis procera (Ait.) R. Br. and Calotropis gigantea R. Br. are the two common species in the genus. Morphologically both C. procera (1-4 m) and C. gigantea (> 4.5 m) are erect shrubs with thick, sub-sessile, obovate leaves having a cordate base. The stems and leaves have a waxy appearance and contain milky latex. Flowers are 5 lobed, medium-sized, born on dense, multi-flowered, umbellate cyme arising from the nodes and appearing axillary or terminal. Corolla-lobes (1.2-1.5 cm) in C. procera is erect, white-colored with purple blotches on the upper half and are fragrant, whereas in C. gigantea corolla-lobes (3-3.5 cm) are spreading, uniformly greenish-white or bluish-purple without any fragrance.
The Calotropis sp. has been widely used in the Ayurvedic, Unani, Arabic, and Sudanese -Indian traditional system of medicine for the treatment of various ailments. In ancient ayurvedic medicine, the plant Calotropis procera was known as “Sweta Arka” and Calotropis gigantea as “Shyma Arka”. Both the species are often similar in their botanical aspects and also have similar pharmacological effects. The latex of the C. procera has been shown wound healing activity 10, 11, protection against gastric ulcer 12, anti-inflammatory 13, and antimicrobial activity 14. The flower of C. procera has hepatoprotective activity 15, antipyretic, analgesic, and larvicidal activity 16, 17. Many of the workers have reported antimicrobial activity of C. procera; however, similar reports on C. gigantea is scarce. Thus, the present investigation was aimed at comparative phytochemical screening and assessment of the antimicrobial activity of C. procera and C. gigantea against some human pathogenic bacterial strains.
MATERIALS AND METHODS:
Plant Material: The aerial parts (leaves and young stem) of naturally grown (8-10 years old) C. procera and C. gigantea were used as plant material for phytochemical extraction. The C. procera (Bot-10575) and C. gigantea (Bot-10574) plant materials were collected from R. K. University Anantapuram, Andhra Pradesh (India), and Utkal University campus, Odisha (India) respectively, and herbarium was submitted to the Department of Botany, Utkal University. The collected aerial parts were cleaned under running tap water (15 min) and shade dried at room temperature for 5-7 days. Coarse powder of the dried plant material was prepared mechanically using mortar and pestle.
FIG. 1: FLORAL MORPHOLOGY OF CALOTROPIS PROCERA (A) & CALOTROPIS GIGANTEA (B). A FLOWERING TWIG OF CALOTROPIS PROCERA (C) & CALOTROPIS GIGANTEA (D)
Phytochemical Extraction: Phytochemical extraction was done in the Soxhlet apparatus by a hot continuous extraction method for 48 h 18. Three different solvent systems in the order of increasing polarity such as ethanol (80%), methanol (80%) and distilled water (aqueous) were used sequentially for the extraction process. The extracts so obtained in each solvent were filtered separately using filter paper (Whatman no. 1) and the filtrates were concentrated at 40 °C using a water bath till sticky residue remained in the bottom of the flask. The residue was finally air-dried and stored at 4 °C until bioassay. The stock solutions of the extracts were prepared by dissolving 10mg of dried extract in 10ml of DMSO to get the concentration of 1 mg ml-1 for further experiments.
Phytochemical Screening: The ethanolic, methanolic and aqueous extracts of C. procera and C. gigantea were subjected to qualitative phytochemical screening for phytochemicals such as carbohydrates (Molisch’s test), protein (Biuret test), an amino acid (Ninhydrin test), an alkaloid (Wagner and Dragandreff’s test), terpenoids, flavonoids, glycosides, cardiac glycosides, steroid, phenol, tannin, saponin, and volatile oils 19.
Test for Carbohydrates: To the 2 ml of extract, 2 drops of Molisch’s reagent was added and mixed well. Subsequently 2 ml of conc. H2SO4 was added from the sides of the test tube. The appearance of a reddish violet color ring immediately at the junction of two liquid layers indicated the presence of carbohydrates.
Test for Proteins: Plant extract (2ml) was treated with 2ml of Biuret reagent. The formation of the violet color ring indicated the presence of peptide linkages of the protein molecule.
Test for Amino Acids: Ninhydrin reagent (2ml) was added to 2 ml of crude extract and the solution was kept in a hot water bath for 20 minutes. The appearance of purple color indicated the presence of amino acids.
Test for Alkaloids: The extract (2ml) was acidified by dilution with 1% HCl. The acid layer was used for testing the alkaloids. The acid layer was treated with a few drops of Mayer’s reagent. The formation of creamy white precipitation indicates the presence of alkaloids.
Test for Terpenoids: To the 2 ml of extract, 2 ml of chloroform and 3 ml of conc. H2SO4 was added. The reddish-brown coloration at the interface indicated the presence of terpenoids.
Test for Flavonoids: To the crude extract 5ml of dilute ammonia solution and conc. H2So4 was added. A yellow coloration confirms the presence of flavonoids, which disappears on standing.
Test for Glycoside: To the 2 ml of extract, 1-2 ml of ammonium hydroxide was added and shaken well. The appearance of cherish red color indicates the presence of glycosides.
Test for Cardiac Glycosides: To the 5ml of extract, 2 ml of glacial acetic acid and one drop of ferric chloride solution was added. The layer formed was under-layed with 1ml of con. H2SO4. A brown ring of the interface indicated a deoxy sugar characteristic of cardenolides. A violet ring might appear below the brown ring, whereas in the acetic acid layer, a greenish ring might form just gradually throughout the thin layer.
Test for Steroids: Crude plant extract was mixed with 2 ml of chloroform and conc. H2SO4 was added sidewise. The appearance of red color in the lower chloroform layer indicated the presence of steroids.
Test for Phenols: To the 2 ml of extract, 3 ml of ethanol and a pinch of FeCl3 was added. The formation of greenish-yellow color indicated the presence of phenols.
Test for Coumarins: Extract was dissolved in hot water. After cooling, the solution was divided in two test tubes. To one of the test tube, 10% (w/v) ammonium hydroxide was added, and the other test tube was used as control. The fluorescent color indicated the presence of coumarins.
Test for Tannins: To the 5ml of extract, a few drops of 1% of lead acetate were added. The appearance of a yellow precipitate indicated the presence of tannins.
Test for Saponins: The extract was diluted with 20 ml of distilled water and agitated in a graduated test-tube for 15 min. The formation of a 1cm thick foam layer indicated the presence of saponins.
Test for Volatile Oil: To the 2 ml of extract, 0.1 ml of dilute sodium hydroxide and few drops of diluted HCl were added. The formation of a white precipitate indicated the presence of volatile oils.
Bacterial Strains and Culture Media: For the assessment of antimicrobial activity of plant extracts, two Gram-positive human pathogenic bacteria such as Streptococcus mutans (MTCC-497T) and Bacillus circulans (MTCC-490T) and two Gram-negative bacteria such as Salmonella enteric typhimurium (MTCC-98), and Vibrio cholera (MTCC-3906) were procured from Institute of Microbial Technology (IMTECH) Chandigarh, India. All the bacterial strains were freshly cultured in nutrient broth according to the standard microbiological method 20.
Working Solution of Extracts and Standard Drug: The stock solution (1mg ml-1) of ethanolic, methanolic and aqueous extracts was serially diluted with DMSO to the working solution concentration of 250 µgml-1. The antibiotic Ciprofloxacin (250 µgml-1) was used as a standard drug against the test of bacterial strains.
Agar Well Diffusion Assay: The agar well diffusion assay 21 was carried out to determine the growth inhibition of bacteria by plant extracts. The nutrient broth (Hi-Media, India) was used for broth culture and nutrient agar (Hi-Media, India) was used for the preparation of agar Petri plates (90 mm diameter). The bacterial suspensions were standardized by adjusting the optical density to 0.1 at 600 nm 22 in the UV-VIS spectrophotometer to get a cell suspension of about 1.5×108 CFU/ml. The bacterial inoculum of 100µl was seeded on the surface of the sterile agar plate aseptically and distributed evenly using sterilized glass spreader. Wells of 6.0mm diameter each were prepared on the inoculated plate by sterilized cork borer. Into the agar wells, 50µl of plant extracts and standard drug (250 µg ml-1) were introduced and allowed to stand for 10 minutes for diffusion. Thereafter the plates were incubated at 37 °C for 24 h and examined for zones of inhibition (ZI). The diameter of the zone of inhibition formed by each agent was measured using an Antibiotic Zone Scale (Hi-Media) and the values recorded were expressed to the nearest millimeter. The average diameter of the zone of inhibition of more than 10 mm was considered as effective. Each extract was replicated four times.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): Minimum inhibitory concentration (MIC) was determined for those extracts that showed high efficacy (ZI ≥ 10mm) against microorganisms by the agar well diffusion assay. Ciprofloxacin was used as a reference drug. MIC is the lowest concentration of antimicrobial agent that inhibits visible growth of the microbes, was determined by microbroth dilution test 23. Working solutions of extracts for MIC and MBC were achieved by two-fold serial dilution with DMSO to obtained different concentrations ranging from 1000µg ml-1 to 3.9 µg ml-1 and 1500 µg ml-1 to 1.46 µg ml-1.
Each test tube was filled with 5 ml of nutrient broth, 10µl of bacterial suspension and 50µl of antimicrobial agents of different concentrations. Positive control contains inoculums with no antimicrobial agents whereas, DMSO used as a negative control with no microbes. The test tubes were incubated at 37 °C for 16-20 h. After incubation MIC was determined to be where growth was no longer visible by assessment of turbidity with optical density readings at 595 nm with a Beckman DU-70 UV-Vis Spectrophoto-meter.
The minimum bactericidal concentration (MBC) was defined as the lowest concentration of antimicrobial agent at which 99.9% of the bacteria were killed 24. MBC was determined by transferring 20μl of each culture medium from the test tube (with no visible growth) from the MIC assay and thereafter inoculating into nutrient agar plates. The inoculated plates were then incubated aerobically at 37 °C for 16–20 h for any colony-forming unit (CFU). The concentration of extract where no CFU appeared on petriplate is considered as MBC.
RESULTS AND DISCUSSION:
Phytochemical Screening: The qualitative phytochemical screening of ethanolic, methanolic and aqueous extracts of C. procera and C. gigantea revealed the presence of 14 pharmaceutically active plant metabolites namely carbohydrates, protein, amino acid, alkaloids, terpenoids, flavonoids, glycosides, cardiac glycosides, coumarin, steroid, phenol, tannin, saponin and volatile oils Table 1. The primary plant metabolites such as carbohydrates, protein and amino acid were detected in all the three extracts such as ethanolic, methanolic and aqueous extracts of both C. procera and C. gigantea. Among the secondary plant metabolites, glycosides and cardiac glycosides were detected in all the three extracts of both the Calotropis sp. The other secondary metabolites such as alkaloid, flavonoids, coumarin, steroids, phenols, and tannins were detected in ethanolic and methanolic extracts but were not detected in aqueous extracts of both the species. The terpenoids, saponin and volatile oils were detected only in ethanolic extracts.
Extraction is a critical step for recovering and isolating phytochemicals from plant materials. Extraction efficiency is affected by the chemical nature of phytochemicals, the extraction method used, sample particle size, the solvent used, as well as the presence of interfering substances 25. For the extraction of biomolecules from plants, the solvents used are chosen on the basis of the polarity of the solute of interest. A solvent of similar polarity to the solute may properly dissolve the solute. Multiple solvents can be used sequentially to limit the amount of analogous compounds in the desired yield 26.
According to previous reports, methanol and ethanol can dissolve polar compounds, such as sugar, amino acid, glycoside 27, phenolic compounds with low and medium molecular weights having medium polarity 28, flavonoid 29, terpenoid, saponin and tannin 30. Ethanol has been known as a good solvent for phenol extraction, whereas methanol has been generally found to be more efficient in the extraction of lower molecular weight phenols 31.
TABLE 1: COMPARATIVE PHYTOCHEMICAL SCREENING OF C. PROCERA AND C. GIGANTEA
|Phytochemicals||Ethanolic extract||Methanolic extract||Aqueous extract|
|C. procera||C. gigantea||C. procera||C. gigantea||C. procera||C. gigantea|
[(+) indicates presence, (-) indicate absence]
Based on preliminary phytochemical assay it can be concluded that high molecular weight compounds were extracted through high polarity solvent like water, whereas small and medium molecular weight compounds extract through less polar solvents like methanol and ethanol. Generally, most of the primary and secondary metabolites in plant samples are polar in nature with characteristic small to medium molecular weight; thus most of the phytochemicals of Calotropis sp. were extracted in ethanol and methanol. Further, ethanol (80%) was used as a least polar solvent in the first step of the extraction process; thus, ethanolic extract of C. procera and C. gigantea contained a majority of the secondary metabolites present in their aerial plant parts. Qualitative phytochemical analysis indicated that the phytochemical constituents of C. procera are similar to that of C. gigantea.
Antimicrobial Activity: For the qualitative evaluation of the antimicrobial activity of C. procera and C. gigantea, the ethanolic, methanolic and aqueous extracts (250 µg ml-1) were tested against two Gram +ve and two Gram -ve human pathogenic bacterial strains and antimicrobial activity was compared with that of standard drug Ciprofloxacin (250 µg ml-1). The antimicrobial activity was assessed in terms of zone of inhibition (ZI) formed by agar well diffusion assay and the findings were presented in Table 2. The study revealed variable antimicrobial activities of the plant extracts against the tested bacterial strains. It was recorded that all the three extracts of C. procera and C. gigantea showed highest anti-bacterial activity against S. enterica typhimurium and V. cholera, followed by B. circulans. The least antimicrobial activity of the plant extracts was against the bacterial strains S. mutans. Comparatively, C. procera extracts were showing higher antimicrobial activity than C. gigantea. The antimicrobial efficiency of the plant extracts was in the order of ethanolic > aqueous > methanolic extracts.
TABLE 2: COMPARATIVE ANTI-BACTERIAL ACTIVITY* ASSESSMENT OF ETHANOLIC, METHANOLIC AND AQUEOUS EXTRACTS OF C. PROCERA AND C. GIGANTEA AGAINST HUMAN PATHOGENIC BACTERIAL STRAINS BY AGAR WELL DIFFUSION ASSAY
|Bacterial strains||Reference antibiotic||Ethanolic extract||Methanolic extract||Aqueous extract|
(Mean ± SDE) pooled from 4 independent experiments (n=4) * determined as Zone of Inhibition: ZI (mm). Mean values within the column with the same superscript alphabets are not significantly different (p≤ 0.05) using Duncan’s new multiple range test.
The antimicrobial activity of C. procera and C. gigantea was quantitatively evaluated in terms of MIC and MBC Table 3. The concentration range of MIC and MBC for different plant extracts varied from 46.87 - 375 µgml-1. For an ethanolic and aqueous extract of C. procera the lowest value of MIC (46.87 µgml-1) was against S. enteric typhimurium and V. cholera. However, lowest value of MBC for ethanolic extract was against S. enteric typhimurium (46.87 µgml-1) and that of aqueous extract was against V. cholera (93.75 µgml-1).
For a methanolic extract of C. procera lowest value of MIC and MBC was against V. cholera (62.5 µgml-1). For C. gigantea lowest value of MIC and MBC of ethanolic extract (62.5 µgml-1) and methanolic extract (93.75 µgml-1) was against both S. enteric typhimurium and V. cholera. For aqueous extract of C. gigantea lowest value of MIC (46.87 µgml-1) and MBC (93.75 µgml-1) was against V. cholera.
The study suggested that ethanolic and aqueous extracts were having lower MIC value than methanolic extracts of C. procera and C. gigantea against the tested bacterial pathogens. Comparatively, the extracts of C. procera were more efficient in bacterial growth inhibition than C. gigantea in terms of MIC and MBC. The bacterial pathogens S. enterica typhimurium and V. cholera were highly sensitive to the plant extracts of C. procera and C. gigantea. The MBC ≤ 125µgml-1 was considered to be very active, and all the extracts were very active against S. enterica typhimurium, V. cholera and B. circulans. However, compared to the reference antibiotic, the extracts of C. procera and C. gigantea have much higher MIC and MBC values.
TABLE 3: MINIMUM INHIBITORY CONCENTRATION (MIC) AND MINIMUM BACTERICIDAL CONCENTRATION (MBC) OF ETHANOLIC, METHANOLIC AND AQUEOUS EXTRACTS OF C. PROCERA AND C. GIGANTEA AGAINST HUMAN PATHOGENIC BACTERIAL STRAINS
|Bacterial strain||Reference antibiotic (µg ml-1)||Ethanolic extracts (µg ml-1)||Methanolic extracts (µg ml-1)||Aqueous extracts (µg ml-1)|
|C. procera||C. gigantea||C. procera||C. gigantea||C. procera||C. gigantean|
S. m: Streptococcus mutans, B. c: Bacillus circulans, S. et: Salmonella enterica typhimurium, V. c: Vibrio cholerae
Data pooled out from 4 independent experiments (n=4); extracts with values in bold font considered as very active (MBC ≤125 μg/ml)
The development of antimicrobial drugs from higher plants plays a central role in the healthcare system over the world 32, 33. There has been an increased interest in the study of medicinal plant extracts as a potential source of new antimicrobial agents. The discovery of such new antimicrobial drugs makes an important field of research, as there is an increase in resistance to existing antibiotics by several pathogenic bacteria 34. The present study revealed that two Gram-negative bacterial pathogens, such as Salmonella enterica typhimurium and Vibrio cholera were highly sensitive to extracts of C. procera and C. gigantea. The gram-negative bacterial pathogen S. enterica typhimurium causes infection in the intestinal tract, typhoid fever, food poisoning and gastroenteritis. Whereas, the V. cholera causes serious diseases like cholera.
The Gram-positive bacteria Bacillus circulans, an opportunistic pathogen known to cause sepsis, wound infection, bacteremia, abscesses and meningitis in immune-compromised persons 35, 36 was also sensitive to all the extracts of C. procera and C. gigantea. However, the Streptococcus mutans, a Gram-positive pathogen reported to cause dental caries 37 is not much sensitive to the plant extracts compared to the other tested pathogens. These findings indicated broad-spectrum antimicrobial activity of the plant extracts against both Gram-positive and Gram-negative bacteria, particularly very effective against Gram-negative enteric pathogens.
Drugs derived from plants are generally secondary metabolites and their derivatives 38. From the phytochemical screening, it was clear that the Calotropis sp. contained numerous bioactive compounds, including alkaloids, steroids, coumarin, flavonoids, glycosides, tannins and saponins. These secondary metabolites act as a defense mechanism against several micro-organisms, insects and herbivorous 39. Phenolic and flavonoids contents in various fruits and vegetables are reported to help immune-modulator organs for killing microorganisms 40. Tannins are polymer of phenolic compounds, found in Calotropis sp. have natural antifungal and antibacterial properties 41, 42. Thus, the antimicrobial activity of the crude ethanolic, methanolic and aqueous extracts of Calotropis sp. was due the presence of secondary metabolites.
As the plant extracts were crude, which contains active ingredients responsible for antimicrobial activity along with other phytochemicals, the MIC and MBC values of plant extracts were much higher than the highly purified reference antibiotic. Thus, the isolation, purification and identification of active principles of crude extracts can lead to novel drug development from Calotropis sp. Further, both in-vitro and in-vivo research is required to evaluate the biological effects of such formulated drugs before application in clinical use.
CONCLUSION: The present investigation supports and validates the antimicrobial aspect of two important ethnomedicinal species C. procera and C. gigantea of the genus Calotropis. The comparative phytochemical screening showed similar phytoconstituents of C. procera and C. gigantea. Several secondary plant metabolites have been qualitatively detected in crude extracts of the plants. The antimicrobial assay indicated higher antimicrobial activity of C. procera than C. gigantea terms of larger zone of inhibition and lower MIC/MBC values. The secondary metabolites detected in the crude extracts of Calotropis sp. responsible for the antimicrobial activity could be a viable alternative source of bioactive compounds with pharmaceutical relevance for the control of antibiotic-resistant human pathogenic bacteria.
ACKNOWLEDGEMENT: Authors acknowledge the University Grant Commission (UGC), New Delhi, for financial support to RS in the form of Rajiv Gandhi National Fellowship (RGNF). Authors thank Dr. T. Ravi Kumar, Department of Botany, R. K. University, Anantapuram, Andhra Pradesh, India, for providing C. procera plant samples.
CONFLICTS OF INTEREST: The authors declare that there are no potential conflicts of interest.
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How to cite this article:
Sethy R and Kullu B: Comparative phytochemical screening and antimicrobial activity of Calotropis sp. of ethnomedicinal significance. Int J Pharm Sci & Res 2020; 11(7): 3243-51. doi: 10.13040/IJPSR.0975-8232.11(7).3243-51.
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