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Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum



S.K. Biswas, A. Chowdhury, J. Das, U.K. Karmakar, S.Z. Raihan, A.C. Das, M.A. Hannan, M.A. Monsur Dinar, M.J. Hassan, M.I. Hossain and M.R. Farhad
 
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ABSTRACT

Cuscuta epithymum Murr. (Convolvulaceae) is found in Bangladesh and has been used in traditional medicine. The present study was designed to investigate the preliminary phytochemical screening, cytotoxicity and antibacterial activities of the methanol extract of the plant stem. The preliminary phytochemical analysis was performed on the basis of the standard procedures and thin layer chromatography to confirm the presence of chemical compounds in the methanol extract of the plant. The minimum inhibitory concentration and cytotoxic activities of the extract were carried out according to broth dilution assay and brine shrimp lethality bioassay, respectively. The results of preliminary phytochemical analysis showed the presence of flavonoid, glycoside, alkaloids, carbohydrates, saponins and steroids in the plant extract. In this study, the methanol extract was subjected to Thin Layer Chromatography (TLC) in order to detect the phytoconstituents present in the extract and the best result of TLC was obtained from the solvent system comprising of toluene and ethyl acetate at the ratio of 5.5:4.5 which identified the maximum number of spots. Moreover, the extract showed moderate antibacterial activities against Bacillus megaterium, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi with MIC values of 4.96±0.20, 3.03±0.16, 3.47±0.20 and 4.07±0.08 mg mL-1, respectively. The methanol extract showed lethality against brine shrimp nauplii (LC50: 36.31 μg mL-1 and LC90: 83.18 μg mL-1 ). The results indicated that the methanol extract of the stem of the plant possessed antibacterial and cytotoxic properties with several numbers of chemical compounds.

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S.K. Biswas, A. Chowdhury, J. Das, U.K. Karmakar, S.Z. Raihan, A.C. Das, M.A. Hannan, M.A. Monsur Dinar, M.J. Hassan, M.I. Hossain and M.R. Farhad, 2012. Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum. International Journal of Pharmacology, 8: 422-427.

DOI: 10.3923/ijp.2012.422.427

URL: https://scialert.net/abstract/?doi=ijp.2012.422.427
 
Received: January 30, 2012; Accepted: March 06, 2012; Published: June 13, 2012



INTRODUCTION

The Convolvulaceae is a family of twining, prostate herbs that has about 1000 species in 40 genera, of which only one genus, Cuscuta, is parasitic. The herb is most widely distributed in man’s crops. The flowering and seed production of Cuscuta epithymum continues for several months but it does not require a host stimulant for seed germination. The Cuscuta plant does not grow equally on all plants that it parasitizes. It may grow exceptionally well, may survive, produce seed quickly and die or may simply retain attached and wait for a better host. The herb is known as ‘Swarnalata’ in India and Bangladesh and as ‘Dodder’ in Australia (Holm et al., 1997). Cuscuta epithymum Murr. belongs to the family of Convolvulaceae (Pagani and Ciarallo, 1974). The herb is a weed parasitizing and it is very difficult to kill it (Dimitrova, 2004). Moreover, they are not photosynthetically active and the leaves are very little (Holm et al., 1997).

The phytochemical compounds present in the traditional medicinal plants can be used in the treatment of different types of diseases (Pareta et al., 2011). Cuscuta epithymum possesses mild laxative and diuretic properties. The plant can also be employed for the treatment of sciatica, scurvy and scrofula derma (Holm et al., 1997). It was reported that the onset of action of seizure was delayed by the methanol extract of Cuscuta epithymum stem in pentylenetetrazol induced mice at the dose of 100 mg kg-1. The plant also possessed effective anticonvulsant components (Mehrabani et al., 2007) and some flavonoids (Pagani and Ciarallo, 1974). From the literature survey, it was observed that no extensive research was done on the plant. Due to the lack of scientific data of this medicinal plant, the present study was carried out to investigate the presence of phytochemicals, antibacterial activity and brine shrimp lethality bioassay for cytotoxic activities of the methanol extract of Cuscuta epithymum stem.

MATERIALS AND METHODS

Collection of plant materials and preparation of the extract: The stems of Cuscuta epithymum were collected from Chittagong, Bangladesh and were taxonomically identified by the experts at Bangladesh Forest Research Institute. The voucher specimen was deposited at the Department of Pharmacy of BGC Trust University Bangladesh. About 200 g of the dried powdered material of the plant was weighed and soaked in 600 mL of methanol. The mixture was then filtered and the excess solvent was completely evaporated to make a yellowish residue (yield 19.2%) which was known as crude methanol extract. Finally, the methanol extract was stored in a desiccator before use.

Culture media and test microorganisms: In this study, nutrient agar was used as a media for bacterial growth. Both gram positive bacteria such as Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus and gram negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Shigella dysenteriae, Shigella sonnei, Salmonella typhi and Vibrio cholerae were used in this study. All of the strains were collected from International Centre for Diarrhoeal Disease and Research, Bangladesh.

Standard drug: The standard drug, amoxycillin was collected from Incepta Pharmaceuticals Limited, Dhaka, Bangladesh.

Preliminary phytochemical screening: Qualitative phytochemical tests were performed for the methanol extract of Cuscuta epithymum according to the standard procedures described by Trease and Evans (1989).

Thin layer chromatography (TLC): Thin layer chromatography is a common and most convenient tool for phytochemical analysis of plant constituents, particularly for their initial separation, detection and identification. The methanol extract of Cuscuta epithymum was subjected to thin layer chromatographic analysis to observe the presence of number of chemical constituents to support the phytochemical tests. The thin layer chromatographic analysis was done according to the procedure described by Mendham et al. (2002). Silica gel slurry was used to prepare the TLC plates and the solvent system selected for the TLC of methanol extract of Cuscuta epithymum was toluene and ethyl acetate as a mobile phase with different ratio of 9.5:0.5, 8.5:1.5, 7.5:2.5, 6.5:3.5, 5.5:4.5, 4.5:5.5, 3.5:6.5, 2.5:7.5, 1.5:8.5 and 0.5:9.5.

Cytotoxic activity: The cytotoxic activity of the crude methanol extract was performed on brine shrimp nauplii according to brine shrimp lethality bioassay (Meyer et al., 1982). From this study, LC50 (μg mL-1) and LC90 (μg mL-1) of the crude methanol extract of the plant were also determined.

In vitro antimicrobial assay and minimum inhibitory concentration (MIC): The antimicrobial activity of the extract was carried out using the agar disc diffusion method (Bauer et al., 1966). The concentration of the plant extract was 500 μg disc-1 while the standard drug, amoxycillin was used at 10 μg disc-1. The minimum inhibitory concentration was determined by broth dilution assay with minor modifications (Kubo et al., 1994; Sahin et al., 2003).

Statistical analysis: Three replicates of each sample were used for statistical analysis and the results of the experiment were expressed as Mean±standard deviation (SD).

RESULTS

Phytochemical screening: The results of various chemical tests for the detection and identification of chemical constituents are summarized in Table 1.

Table 1: Results of preliminary screening of methanol extract of Cuscuta epithymum
Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum
+: Present, –: Absent

The phytochemical analysis showed the presence of flavonoid, alkaloids, glycoside, carbohydrates, saponins and steroids while tannins, anthraquinones, resin and protein were not found in the extract.

TLC analysis: The solvent system selected for the TLC of methanol extract of Cuscuta epithymum was toluene and ethyl acetate as a mobile phase with the ratio of 9.5:0.5, 8.5:1.5, 7.5:2.5, 6.5:3.5, 5.5:4.5, 4.5:5.5, 3.5:6.5, 2.5:7.5, 1.5:8.5 and 0.5:9.5 resulted in identification of spots with different Rf values which are shown in Table 2. The study results revealed that a good number of chemical compounds were present in the crude extract. The solvent system having toluene and ethyl acetate of the ratio 5.5:4.5 produced maximum four spots at 365 nm with Rf values of 0.34, 0.671, 0.7968 and 1.00. The TLC plates of the extract of Cuscuta epithymum are shown in Fig. 1.

In-vitro antibacterial activities and minimum inhibitory concentration: The results of antibacterial activities are summarized in Table 3. The antibacterial potentials of the methanol extract of Cuscuta epithymum were carried out against ten bacterial strains and the results were compared with a standard disc of amoxycillin (10 μg disc-1). At 500 μg disc-1, the methanol extract of the plant showed moderate antibacterial activity against four bacterial strains such as Bacillus megaterium (11.63±0.10 mm), Pseudomonas aeruginosa (6.13±0.10 mm), Escherichia coli (9.72±0.21 mm) and Salmonella typhi (9.95±0.07 mm) while Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Shigella dysenteriae, Shigella sonnei and Vibrio cholerae were not susceptible to the tested crude extract.

Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum
Fig. 1: TLC plate of methanol extract of Cuscuta epithymum

Table 2: Solvent system with Rf values of the compounds present in methanol extract of Cuscuta epithymum
Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum

Table 3: Antibacterial activities of methanolic extract of Cuscuta epithymum
Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum
Data were represented as Mean±SD of triplicate determination, -: No inhibition

Table 4: The MIC values of methanol extract of Cuscuta epithymum against the tested microorganisms
Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum
Data were represented as Mean±SD of triplicate determination, -: No inhibition, *Amoxycillin all values were in μg mL-1

Table 5: Brine shrimp lethality bioassay of methanolic extract of Cuscuta epithymum
Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum

The maximum zone of inhibition obtained against gram positive bacterial strain such as Bacillus megaterium (11.63±0.10 mm). Subsequent tests were also carried out to determine the inhibitory concentrations of the methanol extract of Cuscuta epithymum. Table 4 summarized MIC values against Bacillus megaterium, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi were 4.96±0.20, 3.03±0.16, 3.47±0.20 and 4.07±0.08 mg mL-1, respectively. The obtained MIC values were also compared with the standard drug, amoxycillin.

Cytotoxic activities: The results of cytotoxic activities are presented in Table 5. The extract showed positive result having LC50 and LC90 values of 36.31 μg mL-1 and 83.18 μg mL-1 by brine shrimp lethality bioassay testing which indicated significant cytotoxic activity of the plant extract. The values were calculated from the best-fit line slope (Fig. 2).

Image for - Phytochemical Investigation and Chromatographic Evaluation with Antimicrobial and Cytotoxic Potentials of Cuscuta epithymum
Fig. 2: Graphical presentation of LC50 (μg mL-1) and LC90 (μg mL-1) of methanol extract of Cuscuta epithymum

DISCUSSION

No extensive research was done on the antibacterial and cytotoxic activities of the methanol extract of Cuscuta epithymum. The present study results showed the presence of flavonoid, glycoside, carbohydrates, saponins, steroids. Thin layer chromatography also confirmed the presence of phytochemicals. The presence of natural flavonoids in the medicinal plants could show the antioxidant and free radical scavenging properties (Middleton and Kandaswami, 1992; Okwu and Orji, 2007). Moreover, tannins are useful in urinary tract infections (Agbafor et al., 2011).

It has also been reported that flavonoids and steroids possess anti-inflammatory (Hossinzadeh et al., 2002; Zakaria et al., 2006; Okokon et al., 2008; Garg and Paliwal, 2011) and analgesic activities (Ramaswamy et al., 1985). Alkaloids also show the inhibition of pain perception (Rahman et al., 2011). Amabeoku and Kabatende (2011) also reported that saponins present in the medicinal plant show the analgesic activity. Moreover, the presence of glycosides in the plant may contribute hypoglycemic effect (Okontak and Aguwa, 2007). Thus, the plant might possess the promising hypoglycemic, analgesic and anti-inflammatory effects which should be investigated. The present study also revealed the antibacterial and cytotoxic activities of the methanol extract of the plant stems. Out of ten bacterial strains, Bacillus megaterium (11.63±0.10 mm), Pseudomonas aeruginosa (6.13±0.10 mm), Escherichia coli (9.72±0.21 mm) and Salmonella typhi (9.95±0.07) were susceptible to the crude methanol extract of the stems. The lowest MIC value was obtained against Pseudomonas aeruginosa (3.03±0.16 mg mL-1). In addition to this, the study also confirmed the cytotoxic activities of the plant having LC50 and LC90 values of 36.31 μg mL-1 and 83.18 μg mL-1, respectively. Thus, the plant may be a potential source of new antibacterial and anticancer drug.

CONCLUSION

The results of this study indicate that the plant is potentially a good source of antibacterial and anticancer drugs. The broad spectra of activity of both plants extract is promising and the isolation of active constituents present in the plant can be the subject for the future scientists.

ACKNOWLEDGMENTS

The authors are thankful to Professor Dr Samir Kumar Sadhu, Pharmacy Discipline of Khulna University, Bangladesh to provide all supports in progress of the research works. We also express our gratitude to the authority of International Centre for Diarrhoeal Disease and Research, Bangladesh and Incepta Pharmaceuticals Ltd., Bangladesh.

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