Antimicrobial activity of the methanolic bark extract of

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Antimicrobial activity of the methanolic bark extract of Holarrhena pubescens (Buch. Ham), its fractions and the pure compound conessine a

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Bina S. Siddiqui , Syed Tahir Ali , Ghazala H. Rizwani , Sabira a

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Begum , Saima Tauseef & Aqeel Ahmad

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H.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan b

Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan c

Department of Microbiology, University of Karachi, Karachi-75270, Pakistan Version of record first published: 11 Aug 2011.

To cite this article: Bina S. Siddiqui, Syed Tahir Ali, Ghazala H. Rizwani, Sabira Begum, Saima Tauseef & Aqeel Ahmad (2012): Antimicrobial activity of the methanolic bark extract of Holarrhena pubescens (Buch. Ham), its fractions and the pure compound conessine, Natural Product Research: Formerly Natural Product Letters, 26:11, 987-992 To link to this article: http://dx.doi.org/10.1080/14786419.2010.537268

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Natural Product Research Vol. 26, No. 11, June 2012, 987–992

Antimicrobial activity of the methanolic bark extract of Holarrhena pubescens (Buch. Ham), its fractions and the pure compound conessine


Bina S. Siddiquia*, Syed Tahir Alib, Ghazala H. Rizwanib, Sabira Beguma, Saima Tauseefc and Aqeel Ahmadc a H.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan; bDepartment of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan; cDepartment of Microbiology, University of Karachi, Karachi-75270, Pakistan

(Received 28 October 2009; final version received 20 October 2010) The antimicrobial activity of the methanolic extract of the bark of Holarrhena pubescens, its fractions, and conessine, a steroidal alkaloid, was determined against various bacteria and fungi using the agar diffusion method. They were all found to possess significant activity against some of the bacteria tested. The alkaloidal fraction and conessine also exhibited marginal activity against some of the fungi tested. The minimum inhibitory concentration (MIC) value of conessine was determined against various bacteria, and the highest activity was seen against Micrococcus luteus ATCC 9341 (MIC: 15.6 mg per disc). Keywords: Holarrhena pubescens; Apocynaceae; antibacterial activity; antifungal activity; conessine

1. Introduction Holarrhena pubescens (Buch. Ham) (Synonym: Holarrhena antidysenterica) (Endress, Hesse, Nilsson, Guggisberg, & Zhu, 1990) belongs to the family Apocynaceae. It is an Indian medicinal tree 30–40 ft. high and up to 4 ft. in girth. The tree is common in the forest of India and Pakistan, indigenous to the tropical Himalaya and Assam. The stem bark of this plant, commercially known as ‘kurchi’ has astringent, antidiarrhoeal, antidysenteric, anti-anthelmintic, stomachic, febrifugal, digestive and tonic properties. The bark is used for the treatment of piles, colic, dyspepsia, chest affections and diuresis. It is also used for the treatment of diseases of the skin and spleen. A hot decoction is used as a gargle in toothache. The seeds are effective in diarrhoea, fever, jaundice and for bladder stones. The main alkaloid conessine has been used for the treatment of amoebic dysentery and vaginitis. It also retards the growth of the tubercle bacilli (Bhattacharjee, 2004; Daniel, 2006; Dymock, Warden, & Hooper, 1890; Mhaskar, Blatter, & Caius, 2000; Sastri, 1959). Antibacterial activity of various Holarrhena species has been reported against some organisms *Corresponding author. Email: [email protected]

ISSN 1478–6419 print/ISSN 1478–6427 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/14786419.2010.537268 http://www.tandfonline.com


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(Aqil, Ahmad, & Owais, 2006; Chakraborty & Brantner, 1999). Stem bark and seeds of the plant are reported to contain a number of alkaloids such as holamine, kurchamine, holaphyllidine, holaromine, mitiphylline, holadysenterine (Kumar, Singh, Bhandari, Gupta, & Kaul, 2007), and non-alkaloids such as kurchinin (Siddiqui, Usmani, Begum, & Siddiqui, 1993), kurchinicin (Siddiqui, Usmani, & Begum, 1994), holarrhenol (Ali & Gupta, 1994). Infectious diseases are the world’s leading cause of premature deaths (Beale, 2004), and emergence of multiple drug resistance in many pathogenic bacteria further add to the seriousness of such diseases. The discovery and development of potent and non-toxic antimicrobial agents has thus become an important scientific challenge. Antimicrobials of plant origin offer enormous therapeutic potential for the treatment of infectious diseases, also mitigating many of the side effects that are often associated with synthetic antimicrobials. In this study, methanolic extract of H. pubescens, its fractions, and conessine, the steroidal alkaloid, were evaluated for their antibacterial and antifungal activities.

2. Results and discussion 2.1. Antibacterial activity Activity of the crude methanolic extract (HP) of the bark of H. pubescens and its various fractions were studied against various Gram-positive and Gram-negative bacteria. The extract (HP) was found to be significantly effective against 4 out of 13 Gram-positive and 1 out of 15 Gram-negative bacteria tested at 1000 mg per disc concentration. Further separation of this extract through the classical method yielded five fractions (HP-B, HP-EA, HP-N-PE, HP-N-E and HP-NEA; Supplementary Scheme S1 – online only). They were found to be active against several Gram-positive and Gram-negative bacteria at this concentration (Supplementary Table S1 – online only). Alkaloidal fraction HP-B showed significant activity against 14 Gram-positive and 14 Gram-negative bacteria. This fraction, on further separation according to the method reported earlier (Siddiqui & Pillay, 1932), yielded conessine as the main alkaloid. The structure of conessine was identified by comparing the spectral data (1H- and 13C-NMR) with those reported in the literature (Zirihi, Grellier, GuedeGuina, Bodo, & Mambu, 2005). It was found to be significantly active against 12 Gram-positive and 4 Gram-negative bacteria (Supplementary Table S1 – online only) at the same concentration. MIC of conessine was also determined against various bacteria, which indicated that it possesses highest activity against Micrococcus luteus ATCC 9341 (MIC: 15.6 mg per disc) (Table 1). It may be noted that conessine was more effective than the total extract and any of the fractions (Supplementary Table S1 – online only) against most of the organisms, suggesting that it is the principal active constituent of the bark.

2.2. Antifungal activity The in vitro antifungal activity of methanolic extract (HP) and its various fractions (HP-B, HP-EA, HP-N-PE, HP-N-E and HP-NEA; Supplementary Scheme S1 – online only) and conessine were determined against four filamentous fungi, six dermatophytes and four other fungi (Supplementary Table S2 – online only).

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Table 1. Minimum inhibitory concentrations.

N

H

H

H

H3C


N CH3

Conessine MIC (in mg per disc) Bacteria tested Gram positive Bacillus cereus Bacillus subtilis Bacillus thuringiensis Staphylococcus epidermidis Staphylococcus saprophyticus Staphylococcus aureus Staphylococcus aureus AB 188 M. luteus M. luteus ATCC 9341 Corynebacterium hoffmanii Corynebacterium xerosis Streptococcus faecalis Gram negative Escherichia coli Salmonella typhi ATCC 13311

Streptomycin

Conessine

0.78 0.78 0.78 0.78 0.19 0.19 0.39 0.78 0.39 0.39 0.39 ND

1000.0 62.5 31.25 125.0 125.0 125.0 125.0 31.25 15.6 62.5 62.5 1000.0

12.5 3.12

1000.0 62.5

Note: ND Not determined.

The alkaloidal fraction HP-B showed activity against 2 out of 14 fungi, while conessine isolated from this fraction was found significantly active against 5 out of 13 fungi tested.

3. Experimental 3.1. Plant material The bark of H. pubescens (H. antidysenterica) was supplied by the courtesy of Hamdard Foundation Pakistan Ltd. It was identified by Miss Ashreen Jahan, a botanist at Hamdard Foundation Pakistan Ltd. A voucher specimen (KUH. no. 67985) has been deposited in the Herbarium of Department of Botany, University of Karachi.

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3.2. Extraction and Isolation Uncrushed bark (10 kg) of H. pubescens (H. antidysenterica) was macerated overnight with 10% methanolic NaOH and repeatedly percolated with MeOH for 48 h (five times) at room temperature in order to hydrolyse the tannates. Each extract was neutralised with 30% aqueous HOAc. The syrupy concentrate (HP) obtained on the removal of solvent from the combined extracts under reduced pressure was acidified by adding 10% aqueous HOAc, and extracted out with EtOAc. The aqueous phase was basified with 20% NH4OH and again shaken out with EtOAc. The moist EtOAc phase was treated with a vigorous stream of CO2 (Siddiqui & Pillay, 1932). The precipitate containing the carbonate bases was filtered and the filtrate was dried over anhydrous Na2SO4 and freed of the solvent under reduced pressure. The residue (20 g) from the filtrate was divided into petroleum ether soluble- and petroleum ether-insoluble fractions. The petroleum ether-soluble fraction yielded conessine (9 g; 0.09%), according to the reported isolation procedure (yield 0.1%) (Siddiqui & Pillay, 1932). The main ethyl acetate phase was washed, dried (Na2SO4) and freed of the solvent under reduced pressure. The residue (HP-EA) thus obtained was divided into petroleum ether-soluble (HP-N-PE) and petroleum ether-insoluble fractions. The petroleum ether-insoluble fraction was further divided into ether-soluble (HP-N-E) and ether-insoluble portions. The ether-insoluble fraction was dissolved in ethyl acetate (HP-N-EA) and the ethyl acetate-insoluble darkish minor material was discarded. The fractions HP-B, HP-EA, HP-N-PE, HP-N-E and HP-N-EA along with methanolic extract (HP) and conessine (Supplementary Scheme S1 – online only) were tested for their antibacterial and antifungal activities. Conessine: Colourless needles (acetone) m.p. 125–126 C (Siddiqui & Pillay, 1932); H-NMR (500 MHz, CDCl3) : 5.32 (1H, m, H-6), 3.03 (1H, d, J ¼ 10.7 Hz, H-18a), 2.44 (1H, m, H-20), 2.33 (6H, s, Me-23 and Me-24), 2.25 (3H, s, Me-22), 2.18 (1H, m, H-3), 1.93 (1H, m, H-18b), 1.07 (3H, d, J ¼ 6.3 Hz, Me-21), 0.90 (3H, s, Me-19); 13 C-NMR (125 MHz, CDCl3) : 141.2 (C-5), 121.2 (C-6), 64.9 (C-3), 63.5 (C-20), 63.4 (C-18), 55.6 (C-14), 53.7 (C-17), 50.6 (C-13), 49.8 (C-9), 41.2 (C-23, C-24), 41.1 (C-22), 38.5 (C-12), 38.3 (C-1), 36.8 (C-10), 34.9 (C-4), 33.6 (C-8), 31.7 (C-7), 27.5 (C-16), 24.7 (C-2), 24.5 (C-15) and 22.1 (C-11); HREIMS: m/z 356.5869 [Mþ], (Calcd for C24H40N2, 356.5878). All data are in agreement with the published data (Zirihi et al., 2005). 1

3.3. Determination of antibacterial activity The disc diffusion method (Bauer, Kirby, Sherris, & Turck, 1966) was used to determine the antibacterial activity of the samples. A total of 100 mg mL1 of stock solution in DMSO of each sample was prepared. Sterile filter discs containing 10 mL of stock solution were used for screening. The Iso sensitest agar (Oxoid) plates were seeded with 24-h old culture grown in Mueller Hinton broth (Oxoid). The prepared discs were placed onto the agar surfaces at different positions and plates were incubated at 37 C for 24 h. Results were recorded by measuring the zone of inhibitions in mm. DMSO was used as negative control (Supplementary Table S1 – online only).

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3.4. Determination of MIC MIC of conessine was determined by the disc diffusion method of Bauer et al. (1966). Sterile discs containing different concentrations of conessine varying from 0.98 to 1000 mg per disc were prepared. Streptomycin sulphate (Wuhan pharma factory China) was used as positive control for antibacterial activity. The concentrations of antibiotic used were varied between 0.098 and 200 mg per disc. The MIC was determined as the lowest concentration of conessine showing the zone of inhibition at 10 mm (Table 1).


3.5. Determination of antifungal activity Antifungal activity was also determined by the disc diffusion method (Bauer et al., 1966) as above. Briefly, a small amount of culture was transferred to 2–3 mL distilled water or normal saline in a screw-capped tube with a few glass beads (1 mm in diameter) and vortexed for 5–10 min to make a homogeneous suspension of fungal culture. Sabouraud dextrose agar (SDA) plates were seeded with this suspension. Sterile filter discs containing 1000 mg per disc of sample were placed onto the surfaces at different positions. Plates were incubated at room temperature for 1 week. Results were recorded by measuring the zone of inhibitions in mm (Supplementary Table S2 – online only).

Supplementary material Scheme S1 and Tables S1 and S2 relating to this article are available online.

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