Antibacterial activity of chemical constituents isolated from Asparagus racemosus

  • Muhammad Abdullah Shah Department of Pharmacy, Sarhad University of Science & Information Technology, Peshawar, Pakistan
  • Syed Muhammad Abdullah Department of Computer Science, Islamia College University, Peshawar, Pakistan and Institute of Biological and Chemical Sciences, Peshawar, Pakistan
  • Muhammad Azim Khan Department of Weed Science, KPK Agricultural University, Peshawar, Pakistan
  • Gulfam Nasar NCE in Physical Chemistry, University of Peshawar, Peshawar, Pakistan
  • Irum Saba Department of Pharmacy, University of Peshawar, Peshawar, Pakistan
Keywords: Antibacterial, Asparagus racemosus, E. coli, S. aureus
DOI: 10.3329/bjp.v9i1.16672

Abstract

Asparagus racemosus is a medical extensively used in traditional medicine for various disorders including its use in infectious. So far, work has been done to identify its active constituents responsible for antiseptic folk use of this plant. In the current investigation, we have made an effort to identify its chemical constituents that might be partly responsible for antimicrobial properties. Extraction and isolation of plant extract lead to isolation of two nor-lignans and two steroidal triterpenes (compound 1 to 4). All compound showed considerable antibacterial activities against E. coli and S. aureus while no significant activity was observed against S. typhi. This study highlighted the potential of A. racemosus to be further explored as a source of bioactive natural products.

Introduction

Asparagus racemosus is a climbing perennial herb. It has folk usage as a treatment for dyspepsia and gastric ulcers as well as being used as a galactogogue. Other folk medicinal properties generally attributed to this plant include emollient, cooling, nerve tonic, constipating, galactogogue, aphrodisiac, diuretic, rejuvenating, carminative, immunostimulant, gastroprotective and antiseptic effects (Patel and Patel, 2013; Ravishankar et al., 2012; Battuand Kumar, 2010). In literature, extensive work has been done on various pharmacological properties like phytoestrogen effects (Sharma et al., 1996), adaptogenic effect (Rege et al., 1999), hypolipidemic effect (Visavadiya and Narasimhacharya, 2005), immunomodulatory effects (Diwanay et al., 2004), antibacterial activity (Mandal et al., 2000; Patel and Patel, 2013; Ravishankar et al., 2012; Battu and Kumar, 2010), antidepressant (Singh et al., 2009; Meena et al., 2011), memory enhancing activity (Ojha et al., 2010), antidiabetic effects (Kanwar et al., 2010; Hannan et al., 2007, 2011), and antiulcer (Bhatnagar et al., 2005; Sairam et al., 2003).

We designed a preliminary study to explore its antibacterial activity of its active constituents. Previously, the plant extract of leaves (Patel and Patel, 2013; Battu and Kumar, 2010) and roots (Ravishankar et al., 2012) has showed considerable antibacterial activity against pathogenic bacteria (Mandal et al., 2000). In the current investigation, we have reported various isolated compounds from A. racemosus that might be at least partially responsible for its effect.

Materials and Methods

Plant

Plant material was collected from Malakand division of the KPK Province, Pakistan, in April 2012. Plant material was dried under shade at room temperature for several weeks. After drying, plant material was subjected to grinding and pulverization, which was then stored at low temperature for further processing.

Extraction and isolation

Powdered plant material (16.2 kg) was macerated in distilled methanol for 72 hours, which was followed by filtration and evaporation at reduced pressure under vacuum using rotary evaporator at 40°C. This process was repeated four times that afforded crude methanolic extract (1.319 Kg). The crude methanolic extract was subjected to liquid-liquid extraction suing various organic solvents to get its successive fractions. After fractionation, n-hexane (112.3 g), dichloromethane (352.7 g), ethylacetate (482.9 g) and aqueous (372.4 g) fractions were obtained. Ethylacetate acetate fraction (350 g) was subjected to column chromatography (CC) using silica gel. Sample was eluted initially with increasing polarity of n-hexane in combination with dichloromethane (DCM) ranging from 10%DCM in n-hexane to 100%DCM. Afterwards, increasing polarity was used by combination of methanol with DCM, ranging from 1%methanol in DCM to 20%methanol in DCM. This process afforded 16 sub fractions (AE1 to AE16). Subfraction AE 7 (3.59 g) was subjected to further purification through CC using si gel. The sample was eluted with 4%methanol:DCM, as a gradient, which afforded compound 1 (142 mg and 2 (229 mg). Another subtraction AE9 was also purified via CC using si gel employing 6% methanol:DCM as a mobile phase, which provided compound 3 (87 mg) and 4 (176 mg). Chemical structures (Figure 1) of the isolated compounds 1 (isoagatharesinol; Yang et al., 2004), 2 (gobicusin A), 3 (asparacosin A; Zhang et al., 2004) and 4 (muzanzagenin; Oketch-Rabah et al., 1997) were identified by comparing their Mass and NMR data with reported in literature.

Figure 1: Chemical structures of the isolated compounds

Antibacterial activity

In order to perform antibacterial activity various bacterial strains were used. Bacterial strains were E. coli, S. aureus, and S. typhi. Bacterial strains were grown and maintained on an agar slant at controlled temperature (4°C). The strains were activated for experiment at 37°C for 24 hours on nutrient agar (NA), before any experimental screening. Antimicrobial tests were carried out as reported earlier (Jan et al., 2009), according to disc-diffusion method. In medium, holes of 6 mm diameter were made on the MHA plate (8 mm thick) and filled with 150 μL of test samples (in different concentrations) or standard drug. The inoculated plates were incubated at 37°C for 24 hours. Antibacterial activity was evaluated by measuring the diameter of the zone of growth inhibition around the hole. The assay was repeated three times and the mean diameter was recorded. The minimum inhibitory concentration (MIC) was determined using the same method. Streptomycin, was used as standard antibiotic for comparison with test samples.

Result and Discussion

Bacterial infections are still posing a threat to humanity in successfully treating patients. New but effective antibacterial agents are very crucial for prompt therapeutic management of bacterial infections. Resistance to currently available drugs is an alarming and needs serious and timely efforts. In the current investigation, various compounds isolated from A. racemosus showed considerable antibacterial activity against selected bacterial strains except S. typhi (Table I). S. aureus was the most susceptible bacterial strains, which is commonly associated with respiratory infections. Compound 2, 3 and 4 showed significant antibacterial activity (Table I) against S. aureus, with MIC values 0.05 mg/mL, while compound 1 showed slightly lower activity against the same pathogen. In case of E coli, compound 2 and 4 showed significant activity with MIC values being 0.12 mg/mL. However, compound 3 showed lowest activity against E. coli. In case of compound 1, moderate activity was observed. This preliminary data indicates promising potential of the plant to be further explored for discovery of new antibacterial agents to control ongoing crisis of merging resistance in clinical management of infectious diseases. Extensive work is required to identify molecular mechanisms involved behind the antibacterial effects of the isolated compound. Structural modifications and further experimental work is warranted to modify their potency and efficacy against bacterial pathogens.

Table I: Antibacterial activity of isolated compounds

Treatment Minimum inhibitory concentration
(mg/mL)
E. coli S. aureus S. typhi
Compound 1 0.25 0.12 n.d
Compound 2 0.12 0.05 n.d
Compound 3 0.5 0.05 n.d
Compound 4 0.12 0.05 n.d
Streptomycin 0.01 0.01 0.015

References

Battu GR, Kumar BM. Phytochemical and antimicrobial activity of leaf extract of Asparagus racemosus Willd. Pharmacognosy J. 2010; 2: 456-63.

Bhatnagar M, Sisodia SS, Bhatnagar R. Antiulcer and antioxidant activity of Asparagus racemosus WILLD and Withaniasomnifera DUNAL in rats. Ann New York Acad Sci. 2005; 1056: 261-78.

Diwanay S, Chitre D, Patwardhan B. Immunoprotection by botanical drugs in cancer chemotherapy. J Ethnopharmacol. 2004; 90: 49-55.

Hannan JM, Khatoon S, Rehana F, Jahan S, Rahman MM, Shelley SH. Antihyperglycaemic activity of Asparagus racemosus roots is partly mediated by inhibition of carbohydrate digestion and absorption, and enhancement of cellular insulin action. Br J Nutr. 2011; 6: 406-16.

Hannan JMA, Marenah L, Ali L, Rokeya B. Insulin secretory actions of extracts of Asparagus racemosus root in perfused pancreas, isolated islets and clonal pancreatic β-cells. J Endocrinol. 2007; 192: 159-68.

Kanwar AS, Bhutani KK. Effects of Chlorophytum arundinaceum, Asparagus adscendens and Asparagus racemosus on pro-inflammatory cytokine and corticosterone levels produced by stress. Phytother Res. 2010; 24: 162-66.

Mandal SC, Nandy A, Pal M, Saha BP. Evaluation of antibacterial activity of Asparagus racemosus Willd. root. Phytother Res 2000; 14: 118-19.

Meena J, Ojha R, Muruganandam AV, Krishnamurthy S. Asparagus racemosus competitively inhibits in vitro the acetylcholine and monoamine metabolizing enzymes. Neurosci Lett. 2011; 503: 6-9.

Ojha R, Sahu AN, Muruganandam AV, Singh GK, Krishnamurthy S. Asparagus recemosus enhances memory and protects against amnesia in rodent models. Brain Cogn. 2010; 74: 1-9.

Oketch-Rabah HA, Dossaji SF, Christensen SB, Frydenvang K, Lemmich E, Cornett C, Olsen CE, Chen M, Kharazmi A, Theander T. Antiprotozoal compounds from Asparagus africanus. J Nat Prod. 1997; 60: 1017-22.

Patel LS, Patel RS. Antimicrobial Activity of Asparagus racemesus Wild From Leaf Extracts. Int J Sci Res Pub. 2013; 3: 1-3.

Ravishankar K, Kiranmayi GVN, Lalitha TM, Priyanka T, Ranjith T, Someswarao SBV, Raju VRK, Divya AV. Preliminary phytochemical screening and in vitro antibacterial activity on Asparagus racemosus root extract. Int J Pharm Chem Biol Sci. 2012; 2: 117-23.

Rege NN, Thatte UM, Dahanukar SA. Adaptogenic properties of six rasayana herbs used in Ayurvedic medicine. Phytother Res. 1999; 13: 275-91.

Sairam K, Priyambada S, Aryya NC, Goel RK. Gastroduodenal ulcer protective activity of Asparagus racemosus: An experimental, biochemical and histological study. J Ethnopharmacol. 2003; 86: 1-10.

Sharma S, Ramji S, Kumari S, Bapna JS. Randomized controlled trial of Asparagus racemosus (shatavari) as lactagogue in lactational inadequacy. Indian Paediatr. 1996; 33: 675-77.

Singh GK, Garabadu D, Muruganandam AV. Antidepressant activity of Asparagus racemosus in rodent models. Pharmacol Biochem Behav. 2009; 91: 283-90.

Visavadiya NP, Narasimhacharya RL. Hypolipidemic and antioxidant activities in Asparagus racemosus in hypercholesteremic rats. Indian J Pharmacol. 2005; 37: 376-80.

Yang CX, Huang SS, Yang XP, Jia ZJ. Nor-lignans and steroidal saponins from Asparagus gobicus. Planta Med. 2004; 70: 446-51.

Zhang HJ, Sydara K, Tan GT, Ma C, Southavong B, Soejarto DD, Pezzuto JM, Fong HHS. Bioactive constituents from Asparagus cochinchinensis. J Nat Prod. 2004; 67: 194-200.

Published
2014-01-04

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Research Articles
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Conflict of Interest
Authors declare no conflict of interest