In vitro callus production and antibacterial activity ... - Semantic Scholar

AsPac J. Mol. 2012 Mol. Biol. Biol.Biotechnol. Biotechnol. Vol. 20 (2), 2012 Vol. 20 (2) : 59-64

Barleria lupulina Lindl propagation and activity

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In vitro callus production and antibacterial activity of Barleria lupulina Lindl Sarmad Moin1*, Sahaya Shibu Babu1, Arumugam Mahalakshmipriya2 Department of Biotechnology. Karpagam University, Coimbatore, India Department of Biotechnology, Dr. NGP Arts and Science College, Coimbatore, India 1

2

Received 29 December 2011 / Accepted 27 April 2012

Abstract. Callus cultures of Barleria lupulina Lindl were established from leaf explants on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP), alone and in various combinations. The maximum number of calli was observed in the combination of IAA and BAP (2.0 mg/l). Calli were extracted with methanol to examine the presence of Phytochemicals and its antibacterial activity. Phytochemical analysis revealed the presence of carbohydrates, cardiac glycoside, glycoside, flavonoids, phenols, tannins and terpenoids. The antibacterial activity of the crude methanol extract of the calli showed activity against the Gram positive bacteria Staphylococcus aureus and Bacillus pumilus. Barleria lupulina Lindl leaf-derived callus extract showed the strongest antibacterial activity, with MIC values of 0.375 mg/ml and 0.500 mg/ml against Staphylococcus aureus and Bacillus pumilus, respectively. Keywords: 6-benzylaminopurine (BAP), Antibacterial activity, Barleria lupulina Lindl, Indole-3-acetic acid (IAA), Minimal inhibitory concentration (MIC).

INTRODUCTION Barleria belongs to the family Acanthaceae and is a large, widespread, polymorphic, pantropical genus of herbs and shrubs comprising some 300 species. Its greatest representation is in Africa (particularly the eastern parts) and Asia, with its greatest centre of diversity in tropical East Africa (Balkwill and Balkwill, 1998). Barleria species exhibit several medicinal properties. For instance, leaves of Barleria cristata have been used traditionally for the treatment of variety of diseases including anaemia, toothache, coughs and as a hypoglycemic agent (Gambhire et al., 2009) and juice of the leaves is used in the treatment of ulcers and fever (Ambasta, 1986). Barleria lupulina Lindl has a strong inhibitory effect against acne-inducing bacteria (Chomnawang et al., 2005). Root decoction or infusions of pounded leaves of Barleria eranthemoides R.Br. is drunk for the treatment of dysentery and taken against infectious diseases (Maregesi et al., 2007). Barleria lupulina Lindl is a small shrub, commonly known as Sornomukhi and distributed in South East Asia. It is well known in Thai folk medicine, as the plant is externally used as an anti-inflammatory against insect bites, snake bites, herpes simplex, herpes zoster and varicella zoster virus lesions and it also has a diuretic effect and anti-amoebic activities (Kanchanapoom et al., 2001; Lans et al., 2001; Sawangjaroen et al., 2006). In preliminary investigations (Suksamrarn, 1986; Byrne et al., 1987; Tuntiwachwuttikul et al., 1998) nine iridoid glucosides have been isolated. Compounds found in the leaves of Barleria lupulina Lindl include bar-

lerin, acetylbarlerin, shanzhiside methyl ester, acetylshanzhiside methyl ester, ipolamiidoside and iridoid glucosides (Lans et al., 2001). Virucidal activity against HSV type 2 strain G, in vitro anti-HSV activity, antimicrobial activity against acne-inducing bacteria, antidiabetic potential, antiulcer activity, isolation of Iridoid Glucosides, the crystal Structure and plant growth-inhibiting properties of 6-O-Acetylshanzhiside Methyl Ester, a neuropharmacological profile, and isolation of nine iridoid glucosides from Barleria lupulina have previously been reported (Chomnawang et al., 2005; Kanchanapoom et al., 2001; Lans et al., 2001; Byrne et al., 1987; Yoosook et al., 1999; Suba et al., 2002; Suba et al., 2004; Wirotesangthong and Rattanakiat, 2006). There are various advantages of a cell culture system over the conventional cultivation of whole plants: useful compounds can be produced under controlled conditions independent of climatic changes or soil conditions; cultured cells would be free of infection from microbes and insects; the cells of any plants, tropical or alpine, could easily be multiplied to yield their specific metabolites; automated control of cell growth and rational regulation of metabolite processes would reduce labor costs and improve productivity; and in addition the organic substances can be extracted from callus cultures (Mohammadreza et al., 2005). In this study in vitro callus production, phytochemical screening, and testing of the * Author for correspondence: Sarmad Moin, Department of Biotechnology, Karpagam University, Coimbatore-641021, India. Email: [email protected].

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AsPac J. Mol. Biol. Biotechnol. Vol. 20 (2), 2012

antibacterial activity of leaf derived callus extract of Barleria lupulina Lindl were conducted.

METHODS AND MATERIALS The fresh matured leaves of Barleria lupulina Lindl were collected from the Tamil Nadu Agricultural University (TNAU) campus, Coimbatore, India. The plant species was identified by the Botanical Survey of India, Southern Circle, Coimbatore; the reference number of this authentication is BSI/SC/5/23/08-09Tech.-1655. Leaves of Barleria lupulina Lindl were washed under running tap water and then surface sterilized using 70% ethanol (V/V) for 15 seconds and with 0.01% HgCl2 (W/V) for 7 minutes, followed by five successive rinses in sterile distilled water. Leaf explants were inoculated in the MS medium (Murashige and Skoog, 1962) supplemented with growth regulators IAA (at a concentration of 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/l) and BAP (0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/l) either individually or in combinations (Table 1 and 2). Sucrose (3%) was added and the pH was adjusted to 5.5-5.8 with either 1N NaOH or 1N HCl. 0.8% agar (extra pure gelling point 32-35ºC, Hi-Media) was added to around 4050 ml of the medium which was dispensed into 120 mm × 65 mm culture vessels before being autoclaved at 1.06 kg pressure for about 20 minutes at 121ºC. Five replicates were used for each treatment and cultured at 22 ± 2ºC in the dark. After 3 weeks, calli were subcultured on MS Basal medium and harvested after 4 weeks. Table 1. Effect of IAA and BAP on callus induction from the leaf explants of Barleria lupulina Lindl, after 4 weeks of culture. MS + Growth regulators (mg/l)

Callusing (%)

IAA

BAP

0.5

-

10±0.57

1

-

18±0.48

1.5

-

23±0.23

2

-

35±0.20

2.5

-

26±0.40

3

-

19±0.28

-

0.5

18±0.30

-

1

24±0.23

-

1.5

32±0.43

-

2

40±0.20

-

2.5

30±0.14

-

3

25±0.14

All values are expressed as mean ± SEM.


4 week old leaf-derived calli were collected and dried in a hot air oven at 40±1ºC for 60 hours. The dried calli were homogenized to a fine powder and used for further extraction. For each gram of dry material 2 ml of methanol solvent was used (Lin et al., 1999) for soxhlet extraction over 24 hours. The extract was filtered using Whatman filter paper number 1 and then concentrated at 40ºC using a Rotary evaporator. The residues obtained were stored in a freezer at −20ºC awaiting further tests. Phytochemical analysis was carried out according to the methods described by Harborne, 1984; Trease and Evans, 1989; Raaman, 2006. Qualitative phytochemical analysis of the crude powder extract of the callus derived from Barleria lupulina Lindl was tested for the identification of phytochemicals. In vitro antibacterial activity was examined in the methonolic extract of leaf derived calli from Barleria lupulina Lindl. Microorganisms were obtained from Kovai Medical Centre and Hospital (KMCH), Coimbatore, India. The organisms were sub-cultured on nutrient agar slants and incubated at 37ºC for 24 hours. The organisms were then stored at 4ºC until needed. Among the six micro-organisms, three were Gram-positive bacteria, Bacillus pumilus, Streptococcus pyrogenes and Staphylococcus aureus, while three were Gramnegative bacteria, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia were tested. All the microorganisms were maintained at 4ºC on nutrient agar slants. The antibacterial activity of methanol extracts from calli was determined by the Agar well diffusion method (Bauer et al., 1966), whereby the organism to be tested was spread uniformly on sterile nutrient agar media, a well prepared in the plates with the help of a cork-borer (0.85 cm), 50 µl of the extract (1 mg/ml in methanol) introduced into the well, and the plates incubated overnight at 37ºC. Microbial growth was determined by measuring the diameter of zone of inhibition. For each bacterial strain, pure solvent (100%) and Chloramphenicol were used as negative and positive controls, respectively. The result was obtained by measuring the zone of inhibition in millimetres. One mililitre of nutrient broth was taken in 10 test tubes for each bacterium to determine the minimum inhibitory concentration (MIC) (Mubarack et al., 2011). Different concentrations of plant extracts, 0.125, 0.250, 0.375, 0.500, 0.625, 0.750, 0.875, 1.0, 1.125 and 1.250 mg/ml, were incorporated into the broth and the tubes were then inoculated with 0.1 ml of inoculums of the respective bacteria (at a concentration of 105 CFU/ml) and kept at 37ºC for 24 hours. Simultaneously, a positive control (Chloramphenicol) also tested in various concentrations (10-150 µg/ ml). The test tube containing the lowest concentration of extract showing reduction in turbidity when compared with the negative control was regarded as representing the MIC of that extract.



Table 2. Effect of IAA in combination with BAP on callus induction from the leaf explants of Barleria lupulina Lindl, after 4 weeks of culture. MS + Growth regulators (mg/l)

Table 3. Phytochemical screening of methanol extract of Barleria lupulina Lindl leaf derived callus. Phytochemical constituents

Methanol Extract

Callusing (%)

Alkaloids (Meyer’s Test)

-

Carbohydrate (Fehling’s Test)

+

Cardiac glycoside

+

Flavonoids

+

Glycoside

+

IAA

BAP

1

0.5

20±0.18

1

1

26±0.12

1

1.5

34±0.15

1

2

35±0.14

1

2.5

38±0.29

1

3

39±0.14

2

0.5

40±0.37

2

1

49±0.17

2

1.5

59±0.20

2

2

70±0.14

2

2.5

60±0.14

2

3

41±0.21

RESULTS AND DISCUSSION After 15 days of incubation, initiation of callus growth from leaf explants was observed on the MS medium supplemented with 6-benzylaminopurine (BAP) in combination with indole-3-acetic acid (IAA). Leaf explants present in the MS medium supplemented with BAP and IAA showed the light browning of a friable callus (Figure 1). Maximum callus induction was observed on MS medium in the presence of a combination of BAP and IAA (2.0 mg/l) (Table 2). Premjet, et al. (2010) reported the optimum response of callus production in Barleria prionitis Linn was found in MS supplemented with NAA (1.0 mg/l) and BA (0.5 mg/l). The primary calli were subcultured twice successively. Analysis of the methanol extract of Barleria lupulina Lindl leaf derived calli showed the presence of carbohydrates, glycosides, cardiac glycosides, phenols, tannins, flavonoids, steroids, and terpenoids, whereas alkaloids, phlobatannins, protein, resin, and saponins were absent (Table 3). Similarly dried plant material from Barleria lupulina Lindl has showed the presence of flavonoids and steroids (Doss et al., 2011), where flavonoids, tannins, saponins, carbohydrates, and amino acids have been shown to be present in extracts of Barleria grandiflora Dalz (Sawarkar et al., 2009). Gupta et al. (1984) stated that glycosides was present in the extract of Barleria priontis Linn. The preliminary phytochemical tests of Barleria lupulina Lindl showed a positive result with Dragenodorff's reagent, the Shinoda's test, Ferric Chloride T.S. and the Libermann-Burchard’s test (Phisutthanan and Phisutthanan, 1999). The antibacterial screening results revealed that methanol extract of in vitro grown Barleria lupulina Lindl leafderived calli produced zones of inhibition against Bacillus

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Phenols

+

Phlobatannins

-

Protein (Millon’s Test)

-

Resins

-

Saponins

-

Steroids

+

Tannins

+

Terpenoids

+

‘+’ – Indicates the presence of phytochemical compound, ‘-’ – Indicates the absence of phytochemical compound. Table 4. Antibacterial activity of methanol extract of Barleria lupulina Lindl leaf derived callus, expressed as zone of inhibition. Zone of inhibition (mm) Organisms

Methanol callus extract

Negative Control

Positive control

Escherichia coli

-

-

28±0.23

Pseudomonas aeruginosa

-

-

25±0.21

Klebsiella pneumonia

-

-

26±0.11

Bacillus pumilus

9±0.11

-

22±0.17

Streptococcus pyogenes

-

-

30±0.11

Staphylococcus aureus

6±0.18

-

23±0.17

pumilus and Staphylococcus aureus measuring of about 9 mm and 6 mm, respectively, but no zone of inhibition against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Streptococcus pyogenes (Table 4). The methanol extract produced a 9 mm zone of inhibition against B. pumilus, which was minimal activity in comparison to the positive standard Chloramphenicol. Moderate activities

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Figure 1. Callogenesis from Barleria lupulina Lindl leaf explant. A. Leaves of Barleria lupulina Lindl. B. Callus initiated on MS medium in combination of BAP and IAA (2.0 mg/l). C. Subcultured callus on MS Basal medium. Scale bar A-C represents 5mm. were observed against Staphylococcus aureus for methanol extract compared to the positive control, whereas no activity was observed from the negative control (methanol). Among the pathogenic bacteria only Gram-positive bacteria Bacillus pumilus and Staphylococcus aureus showed susceptibility to the methanol callus extract of Barleria lupulina Lindl. Methanol callus extract of Barleria lupulina Lindl showed no activity against Gram-negative bacteria. This result was similar to the result of Chomnawanga et al. (2005) who reported the antibacterial activity of Barleria lupulina Lindl leaf extract against Staphylococcus epidermidis measured as MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration). Extracts of Senna alata, Eupatorium odoratum, Garcinia mangostana and Barleria lupulina showed strong inhibitory effects (zone of inhibition

≥15 mm) against Staphylococcus epidermidis and Propionibacterium acnes. A chloroform extract of Barleria lupulina Lindl was active against Bacillus cereus (Voravuthikunchai et al., 2006). Similarly Doss et al. (2011) reported that dried plant material of Barleria lupulina Lindl showed activity against Staphylococcus aureus and Escherichia coli, producing 16 mm and 12 mm zones of inhibition, respectively. In the present investigation the antibacterial activity of leaf callus extracts has been demonstrated for the first time. The MIC of the methanol extract is shown in Table 5. Barleria lupulina Lindl showed the strongest antibacterial activity, with MIC values of 0.375 mg/ml and 0.500 mg/ml, respectively, against Staphylococcus aureus and Bacillus pumilus, whereas the positive control produced MIC values of 20 µg/ml and 10 µg/ml for Staphylococcus aureus and Bacillus



Table 5. Minimum Inhibitory Concentrations of methanol extract of Barleria lupulina Lindl leaf derived callus. Organisms

MIC value of methanol extract (mg/ml)

Positive control (µg/ml)

Escherichia coli

-

30

Pseudomonas aeruginosa

-

50

Klebsiella pneumonia

-

20

Bacillus pumilus

0.500

10

Streptococcus pyogenes

-

30

Staphylococcus aureus

0.375

20

pumilus, respectively. Similarly dried plant material from of Barleria lupulina has showed an MIC value of 0.250 mg/ml against Staphylococcus aureus (Doss et al., 2011). Barleria lupulina leaf-derived callus extract shows strong activity according to Gupta and Saxena (1984) who stated that the judgement of strong activity is based on MIC values between 0.05 and 0.50 mg/ml, moderate activity on values between 0.6 and 1.50 mg/lm and weak activity on values above 1.50 mg/ml. In conclusion, Barleria lupulina Lindl showed maximum callus production from leaf explants on Murashige and Skoog (MS) medium supplemented with the combination of IAA (2.0 mg/l) and BA (2.0 mg/l). Calli were extracted with methanol, and antibacterial screening showed activity against Staphylococcus aureus and Bacillus pumilus. However, the components responsible for the antibacterial activities of Barleria lupulina Lindl extract are currently not known. Therefore, it is suggested that further work should be on the isolation and identification of the antimicrobial compound or compounds.

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