Morpho-phenological and Antibacterial Characteristics of Aconitum spp.

Aconitum species have been traditionally used as ethnomedicine to cure various ailments. The present study reveals the morpho-phenology and antibacterial property of alkaloid extracts of the two Aconitum species. The morpho-phenological characteristics will be helpful for determining the resource availability. Aconitum nagarum is erect type, whereas, Aconitum elwesii is a climber. Aconitum elwesii grows in advance of A. nagarum in terms of growth, flowering and senescence. Towards the end of the year, when the fruits have ripened, the parent tuber dies off. As a result, the daughter tuber becomes independent and in the following spring, takes over the function of the parent tuber. Aconitum nagarum and A. elwesii were found to contain 4-5 aconitine equivalent (AE) mg/g of alkaloid. These alkaloids showed antibacterial activity against different bacterial species including human pathogens, namely, Staphylococcus aureus, Salmonella typhimurium, Bordetella bronchiseptica, Escherichia coli, Bacillus subtilis, Pseudomonas putida, Pseudomonas fluorescence and Xanthomonas campestris. However, the extent of antibacterial activity varied among different bacterial species. The antibacterial activity against S. aureus, B. bronchiseptica, and B. subtilis was bactericidal in nature, whereas, against other tested bacterial species was bacteriostatic. Efficacy of the antibacterial activity of these alkaloids was evaluated by comparing with that of standard antibiotics. Differential localization of the antibacterial principle was observed among the Aconitum species studied.


Introduction
Aconitum comprising about 400 species is a circumboreal arctic and alpine genus that extends into lower latitudes where there is suitable mesic habitat at high elevations along the north-south chains of mountains. The greatest distribution of Aconitum species is in Asia, with smaller groups in Europe and North America (Brink and Wood, 1997). About 28 species have been reported in India from sub-alpine and alpine regions between the altitudes of 2000 -4800 m above mean sea level (Sharma et al,. 1993). Botanical Survey of India reported two species namely, Aconitum nagarum Stapf. and Aconitum elwesii Stapf. from Manipur, India (Sharma et al., 1993;Singh et al., 2002). Aconitum, called Nuishiwon in the local dialect, named after a migratory bird, locally called Nuishi which migrates into Shirui peak and has the same bluish colour of Aconitum flower. The extracts of Aconitum species are traditionally being used at diluted concentrations for curing, fever, gastro-intestinal dysfunction, inflammation, cough and asthma. Moreover, its paste is also used for the external applications such as treatment of neuralgia and other painful infections (Mori and Murayama, 1989). Some Aconitum species are also reported to possess antifungal, insecticidal and rodenticidal properties (Deshmukh and Borle, 1976;Smith and Secoy, 1981). The rhizomes of Aconitum species are used by the tribes of Sikkim, India for curing different ailments (Pradhan and Badola, 2008). Although, the alkaloids of Ranunculaceae family are reported to have antimicrobial properties, there are only a few reports available on antimicrobial activity of alkaloids from Aconitum species (Samy and Gopalakrishnakone, 2008). Ukhrul, the present study site is in one of the five hill districts of Manipur and falls under group 11 (Sub-Group 11 BC 1 ) of Eastern Himalayan Wet Temperate Forest type (Champion and Seth, 1968). Understanding the morpho-phenological characteristics of a medicinal plant is as much important as exploring its biochemical characteristics which inform us about the timing and duration of resource availability in the ecological communities. The species reported from this region have not been explored thoroughly for their clinical potential. With the recent trends showing preference for the traditional medicines, the plant-derived compounds are considered a better alternative. Therefore, in the present study, alkaloids from A. nagarum and A. elwesii were extracted and tested for antibacterial activity against a wide range of bacteria including important pathogens. Study also aimed to determine the mode of their antibacterial action, either bactericidal or bacteriostatic and minimum inhibitory concentration (MIC) against these test resulted in milky appearance due to the formation of alkaloid salt precipitate (Wangchuk, 2004).

Quantification of alkaloid using HPLC
High performance liquid chromatography (HPLC) was performed for the quantification of alkaloids from different samples and compared with a standard curve prepared using aconitine (Sigma-Aldrich, Inc, St. Louis, Mo., U.S.A). The alkaloid residue was dissolved in acetonitrile and was analyzed using HPLC system (PU-980 HPLC pump, UV-975 UV/ Vis detector and Rheodyne injector 7725, Jasco, Tokyo, Japan). Octa-decyl silane (ODS) Hypersil (250 × 4.6 mm internal diameter, Thermo Hypersil-Keystone, PA, USA) was used as the stationary phase. The alkaloid peaks were detected at 235 nm. Flow rate was 1 ml/min and the gradient system consisted of solvent A (0.2%, Trifluoroacetic acid) and solvent B (Tetrahydrofuran). A gradient elution of mobile phase, 20 to 40% solvent B in 20 min followed by decreasing gradient of 40 to 0% solvent B in 5 min, then re-equilibrating the column with 20% solvent B in 5 min was used for resolving the alkaloids in the extract. The alkaloid content (mg/g) was expressed in terms of aconitine equivalent.

Analysis of antibacterial activity
Antibacterial activity of the alkaloid extracts was tested against different bacterial species including certain common human pathogens including Staphylococcus aureus, Salmonella typhimurium (MTCC 98), Bordetella bronchiseptica (NCIM 2267), Escherichia coli (MG 1655), Bacillus subtilis (NCIM 2063), Pseudomonas putida (NCIM 2847), Pseudomonas fluorescence (NCIM 2059) and Xanthomonas campestris using disc diffusion method . In brief, the test bacterium was grown overnight in 10 ml of Luria Bertani broth. The pathogenic bacteria including S. aureus, S. typhimurium, B. bronchiseptica and E. coli were incubated at 37°C and other microbes at 28°C. Cultures were diluted to ~10 5 to 10 6 cfu / ml. A 100 μl of this dilution was spread plated on a Luria Bertani agar plate and the plates were dried under laminar airflow for 20 min. The test was conducted using alkaloid extracts at four different concentrations (12.5, 25, 50 or 100 µg / disc) by diluting from the stock of 10 mg/ml prepared in dimethylsulfoxide (DMSO). DMSO without any extract served as a control. Sterile paper discs (6 mm in dia.) were placed aseptically on the inoculated plate. A 20 µl aliquot of the sample was transferred on the disc and the plates were later incubated overnight at appropriate incubation temperature as mentioned above. The diameter of the zone of inhibition was measured.
Ten different concentrations ranging from 0.001 to 50 µg/disc of standard antibiotics, namely, rifampicin, streptomycin, kanamycin and spectinomycin were used to evaluate the efficacy of aconitum alkaloid for their antibacterial property against above mentioned microbes.
bacteria. Further, the efficacy of antibacterial activity of extracts was also compared with that of several common antibiotics such as rifampicin, kanamycin, streptomycin and spectinomycin. Therefore, as an integrated approach, we carried out the present investigation on the morphophenological and antibacterial characteristics of Aconitum species available in this part of Indo-Himalayan region. To the best of our knowledge this is the first study of its kind elucidating the potential clinical significance of Aconitum from this region.

Morpho-phenology
The field survey was carried out during 2007-2010 on monthly basis. Generally, several specimens of each species were collected after making necessary observations on habit, habitat, and morphological characteristics. Specimen collection and herbarium preparation was done according to the method described by Jain and Rao (1958). Specimens were authenticated by comparing with the deposit at Central National Herbarium, Indian Botanic Garden, BSI Howrah, Kolkata, India, namely Aconitum nagarum Stapf, Sirohee: 19.9.1948, Mukerjee, 3515, andAconitum elwesii Stapf., Sirohee: 19.9.1948, Mukerjee, 3518. Voucher specimens SYM-002201, Aconitum nagarum; SYM-002202, Aconitum elwesii, were also deposited at the herbarium of Department of Life Sciences, Manipur University, Imphal, Manipur, India. Morpho-phenological study was carried out according to the method described earlier (Chowdhury et al., 2006;Sharma, 2000).

Extraction of alkaloids
Aconitum alkaloid was extracted by the method described earlier (Ohta et al., 1997). Dried leaves and roots were ground separately into fine powder using a mixer grinder. The fine powder was homogenized with 1M HCl in the ratio of 1:10 (w/v) in a mortar and pestle and filtered through double-layered cheesecloth. The filtrate was centrifuged at 12,500 g for 20 min at ambient temperature (26 ± 2°C) and the supernatant was adjusted to pH 10 with ammonia solution (25%). The resulting suspension was extracted thrice with equal volumes of chloroform. The organic layer was pooled together and washed thoroughly with distilled water to remove ammonia. The organic phase was dried over anhydrous sodium sulfate to remove traces of water molecules and later evaporated with N 2 gas.

Detection of alkaloid
Mayer's reagent (K 2 HgI 4 ) was prepared by dissolving mercuric chloride (1.36 g) and potassium iodide (5 g) in 100 ml of milli Q water. It was used for the qualitative detection of alkaloid in the herbal extracts (Wangchuk, 2004). One ml of extract was transferred to a petri dish and Mayer's reagent was added. The presence of alkaloid

Analysis of mode of antibacterial activity
To determine the possible mode of antibacterial activity, whether bactericidal or bacteriostatic, twenty random points from the zone of inhibition were re-spotted on a fresh Luria Bertani agar plate without having any antibiotics and observed for growth of any viable colony present, up to 72 h of incubation. No growth from the spot was indicative of bactericidal action, whereas, growth from the spot indicated bacteriostatic action. The experiment was repeated at least twice to confirm the observations.

Determination of minimum inhibitory concentration (MIC)
The minimum inhibitory concentration was determined as described earlier (Devienne and Raddi, 2002). The overnight grown culture was appropriately diluted to obtain ~10 5 -10 6 cfu / ml. A 200 µl of Luria-Bertani (LB) medium was incubated with 10 µl of this diluted culture in 96-well microtitre plate and various concentrations of alkaloids prepared in DMSO (Dimethyl sulfoxide) were added to the wells, separately. DMSO alone was taken as control. The pathogenic bacteria namely S. aureus, S. typhimurium, B. bronchiseptica and E. coli were incubated at 37°C for and other test bacteria at 28°C. Absorbance at 600 nm was measured at different time intervals up to 72 h of incubations using ELISA reader.

Morphological Characteristics of Aconitum spp. in Ukhrul District, Manipur, India
Aconitum nagarum Stapf. The morphological and inflorescence characters of Aconitum nagarum are presented in Fig.1 I, 2 a and b. Roots: paired, tuberous with a length of 1.7-2.5 cm; penetrate up to a depth of 14-20 cm below the soil surface. Stem: erect, with diameter up to 1 cm at the base, reaches up to 1.4 m in height, simple, green in colour, terete, finely pubescent with adpressed reversed hairs. The basal leaves soonwithering, upper scattered and few, lowest petioles up to 49 cm long. They are winged, sulcate and amplexicauline. Lamina: cordate-reniform, 3-partite to the base, glabrous with dimension of 6.5-23 × 4.5-15.5 cm, with 4.5-10 cm deep sinus and 3-lobed, lateral lobes unequally two lobuled: lobules acute, 1-2 dentate, sharply and coarsely dentate. Flowers: terminal and axillary over 20-140 cm long racemes; pedicels up to 1.5 -5 cm long, erect or sub erect, bracts foliaceous. 3 partite; sepals blue, upper sepal helmet shaped, 0.5 -2.8 cm long semi orbicular, lateral sepal 0.4-1.5 cm long, sub orbicular, oblique lower sepals deflexed, 1.1 cm long elliptic sub acute: clawed 1.4 cm long, very slender; carpels 5, oblong, pubescent. Follicles oblong, divergent towards the apex, truncate minutely pubescent. ing perennial, the plants in matured phase generally have one mother root with one to two daughter roots to be the successor of the mother root for the upcoming generation. Tab. 1 displays the phenophases of both the Aconitum species. In both the species, sprouting of the daughter root and seeds starts by March and up to July is the vegetative phase. The plants start flowering from the first week of August and September for A. elwesii and A. nagarum, respectively, flowering starts a month earlier in A. elwesii. With onset of second week of September and November fruiting starts in A. elwesii and A. nagarum, respectively, and last up to November in A. elwesii, whereas, in A. nagarum some of the flowers are still in bloom, with initiation of fruiting phase. Plants of both the species get matured by the month of January and December and subsequently the areal parts of the plants become dry by January and last up to February.
The connection of the daughter tuber with the mother plant is maintained by a usually very short neck through which the food material wanders from the stem into the new tuber and gets deposited there mainly as reserve starch and alkaloids. Therefore, it is these daughter tubers which are principally collected, and they should, of course, not be taken up before they have attained maturity.

Alkaloid detection and quantification
In a qualitative assay, the alkaloid extracts from A. nagarum and A. elwesii turned milky upon addition of Mayer's reagent which confirmed the presence of alkaloid in the samples.
The alkaloid content was determined using HPLC and expressed in terms of aconitine equivalent (AE) mg/g. Seeds: transversely lamellate. Flowering and fruiting occurs between September to December.
Aconitum elwesii Stapf. The morphological and inflorescence characters of Aconitum elwesii are presented in Fig.1 II, 2 a and b. Roots paired tuberous with a length of 1.9-4.3 cm; they penetrate up to a depth of 16-25 cm below the soil surface. Stem scrambling, very long, sparingly branched scabrous or puberulous with diameter up to 0.17-0.3 cm at the base, reached up to 2 m high, simple, green in colour, terete, finely pubescent with adpressed reversed hairs. Leaves cauline, scattered petioled with a length of 3.2-6 cm, pubescent; lamina ovate-cordate or rotund with wide sinus, palmately 3-5 partite; lobes cuneate-ovate, finely toothed. Flowers few to many blue or violet in axillary and terminal racemes or or sub-paniculated, over 3-8 cm long; pedicels recurved up to 1.5-2 cm long. Sepals blue or violet, sparingly hairy; uppermost sepal narrowly helmet-shaped, arched, 0.5-2.8 cm, beaked; upper lateral sepals 0.7-1.1 cm long; lower lateral sepals 0.5-1.2 cm; spur reflexed; petals glabrous. Follicles 5, glabrous; testa of seeds plaited. Flowering and fruiting occurs between August to November.
Phenology of Aconitum spp. The sprouting of root stock and germination of seeds in both the species starts during the month of March and continue their vigorous growth in the later part of the year. Six phenological stages are noted and the same are diagrammatically represented by the lines radiating from different angles of a hexagonal figure for each month in regard to individual species (Tab. 1). Both the species be-  Fig. 3 displays the HPLC profile of standard aconitine as well as alkaloid contents including aconitine in A. nagarum root and A. elwesii root. The standard aconitine peak was observed at a retention time of 10 min. The alkaloid content in A. nagarum root and leaves were 4.3 and 5 AE mg/g, while in A. elwesii root and leaf it was 4.7 and 4.8 AE mg/g, respectively.

Antibacterial activities of alkaloids and possible mode of activity
The antibacterial activity of the alkaloid extract was tested against different bacteria including certain human pathogens, namely, S. aureus, S. typhimurium, B. bronchiseptica and E. coli (Tab. 2). The efficacy of antibacterial potency of aconitum alkaloid was evaluated by comparing with the antibacterial activity displayed by certain standard antibiotics, namely, rifampicin, kanamycin, streptomycin and spectinomycin against some of these test organisms (Tab. 3). For a comparative characterization, the antibacterial activity was categorized as weak, moderate and strong depending upon zones of inhibition (weak: 7-8 mm, moderate: 9-11 mm and strong: >12 mm). Alkaloid extract from A. nagarum root showed moderate to strong level of bacterial inhibition against S. aureus B. bronchisep- Tab  alkaloid from A. nagarum leaf (100 µg/disc) was closer to activity displayed by 0.001 µg/disc of rifampicin, 0.1 µg/ disc of kanamycin, 0.1 to 1 µg/disc of streptomycin and 5 µg/disc of spectinomycin (Tab. 2 and 3).
Alkaloid extract from A. elwesii root showed weak to moderate inhibition to B. bronchiseptica, B. subtilis and X. campestris at the concentration of 100 µg/disc. It showed relatively weak inhibition to S. typhimurium, E. coli and P. fluorescence even at the higher concentration of 100 µg/disc. No inhibition was observed for S. aureus and P. putida. Alkaloid extract from A. elwesii leaf showed higher antibacterial activity as compared to A. elwesii root. The extent of antibacterial activity was moderate to strong against B. bronchiseptica, B. subtilis and X. campestris at the higher concentration of 100 µg/disc. Level of inhibition was weak to moderate at same concentration for P. fluores-tica, B. subtilis, P. putida and X. campestris at the higher concentration of 100 µg/disc. The inhibition was found to be weak in case of S. typhimurium, E. coli and P. fluorescence at the same concentration. Antibacterial activity of alkaloid (100 µg/disc) from A. nagarum root was found to be quite close to the activity displayed by 0.001 µg/disc of rifampicin, 1 µg/disc of kanamycin and streptomycin and 10 µg/disc of spectinomycin (Tab. 2 and 3).
Alkaloid from A. nagarum leaf was found to have comparatively less antibacterial activity than that of alkaloid from A. nagarum root. A weak to moderate level of bacterial inhibition was observed at the higher concentration (100 µg/disc). Interestingly, S. aureus, P. putida and X. campestris were found to be less sensitive to alkaloid extract from leaf, however, the sensitivity of other tested bacteria was almost similar to that of root. Antibacterial activity of showed high potency and other alkaloid samples showed low to medium potency.

Discussion
Aconitum in this part of Indo-Himalayan region is yet to explore fully, researchers like Deb (1961) and Sinha (1996) reported Aconitum under the family Helleboraceae, however in recent study, Singh et al. (2002) reported the plant under the family Ranunculaceae, which is accepted worldwide. Singh et al. (2002) reported Aconitum nagarum and Aconitum elwesii from Manipur and mentioned the flowering and fruiting period to be July-October and July-September for A. nagarum and A. elwesii, respectively. However, in the present study after a detailed investigation and observing the phenophases, it was found that flowering and fruiting period is September-December, for A. nagarum and August-November for A. elwesii respectively, with a slight variation in the period from what Singh has reported. However, Rao (1993) reported the plant from Manipur and mentioned the flowering and fruiting period as April-May for A. nagarum and May-September for A. elwesii this might be misapplied, as April-May being the vegetative stage for A. nagarum, as shown in the phenogram Tab. 5.
The alkaloid content of (4-5 AE mg/g) reported in the present study is in concurrence to what Giri et al. (1997) reported in A. heterophyllum root. The overall antibacterial activity of the alkaloid extracts from A. nagarum root and A. elwesii leaf were found to be high and quite similar. The extent of activity was found to be less in the alkaloid from A. nagarum leaf and A. elwesii root. The pattern observed for alkaloid from A. nagarum was opposite to that of A. elwesii due to higher alkaloid content from A. elwesii leaf than A. elwesii root. Therefore, data showed more lo-cence and P. putida. There was no inhibition observed for S. typhimurium and E. coli (Tab. 2 and 3).
Antibacterial activity displayed by 100 µg/disc alkaloid from A. elwesii root and leaf was quite similar to that displayed by 0.001 µg/disc of rifampicin, 0.1 to 1 µg/disc of kanamycin, 1 µg/disc of streptomycin and 10 µg/disc of spectinomycin ( Tab. 2 and 3).
Among the bacteria tested for sensitivity against different antibiotics, B. bronchiseptica was found to be significantly sensitive to kanamycin, streptomycin and spectinomycin, whereas, B. subtilis was observed to be more sensitive to kanamycin and streptomycin. E. coli was found to be more sensitive to streptomycin and spectinomycin. S. aureus was found to be more sensitive to rifampicin (Tab. 3).
The mode of antibacterial activity (whether bactericidal or bacteriostatic) was also determined for these alkaloid extracts against different bacteria including pathogens (Tab. 4). The alkaloid extracts showed bactericidal effect against S. aureus, B. bronchiseptica and B. subtilis, whereas, for other bacterial species the effect was bacteriostatic even at 100 µg/disc concentration.

Minimum inhibitory concentration (MIC)
A wide variation in the MIC was observed for the bacterial species studied (Tab. 5). The antibacterial potency of alkaloid from Aconitum spp. was categorized as high, where MIC was less than 100; medium, where MIC was 100-250; and low, where MIC was more than 250. The antibacterial potency of all the alkaloid extracts was found to be high against B. bronchiseptica and B. subtilis while low for S. typhimurium, E. coli and P. fluorescence. For S. aureus, the alkaloid extract from A. elwesii leaf showed high potency, whereas, other samples showed medium potency. In case of P. putida, alkaloid extract from A. nagarum root Tab  end of the year, when the seeds have ripened, the parent tuber dies. As a result, the daughter tuber becomes independent and in the following spring takes over the function of the parent tuber. Further, our study established the antibacterial property of alkaloid extracts of A. nagarum and A. elwesii against different bacteria including human pathogens, namely, Bordetella bronchiseptica and Staphylococcus aureus. Present work also shows the species specific differential localization of bioactive compounds in different plant parts, which could be favourable for the plant to survive from the predators. Moreover, the Aconitum alkaloids provide an alternate ethnomedicine having wider spectrum of antibacterial activity against different human pathogens. These alkaloids may also provide clues to find out the mechanism of inhibitory action against different bacteria and to synthesize new potential variants.
calization of bioactive alkaloids in root of A. nagarum and leaf of A. elwesii. Previously, root was reported as a major site of alkaloid accumulation in Aconitum species (Pandey et al., 2005), however in the current study, leaf of an Aconitum species was found to contain potent bioactive alkaloid. The antibacterial activity is known to vary significantly among different plant parts (Al-Bayati and AL-Mola, 2008). Interestingly, the Gram-negative bacteria, S. typhimurium, E. coli and P. fluorescence were found to be comparatively less susceptible to the most of the alkaloid extracts tested than the Gram-positive bacteria, S. aureus and B. subtilis. Similarly, total alkaloids extract from Mitragyna inermis have been found to posses comparatively higher antibacterial activity against Gram-positive bacteria than that of Gram-negative bacteria (Zongo et al., 2009). A higher resistance in the Gram-negative bacterial species may be due to the presence of outer membrane, which acts as barrier to the penetration of several antibiotics and the enzymes present in the periplasmic space, which degrades exogenous molecules (Angenot et al., 1991;Tanaka et al., 2006). However, sensitivity of some of the tested Gramnegative bacteria to the alkaloid extracts indicated the wide spectrum of antibacterial activity for these alkaloids. Some alkaloids from Aconitum species are known to cause toxic effects at higher concentrations (Bisset, 1981). Similarly, alkaloids from other plants are also reported to be toxic to vertebrates and insects, and can also inhibit the growth of bacteria and plant seedlings (Wink and Latz-Brüning, 1998). Moreover, the presence of such broader activity spectrum alkaloids in plants may be meant for their multipurpose defense (Wink and Latz-Brüning, 1998). Though the mechanism for antibacterial activity of alkaloids is not yet known. Recent study has shown that alkaloids could inhibit DNA synthesis through topoisomerase inhibition and DNA intercalation (Karoul et al., 2005). Among the human pathogen tested all showed bactericidal in nature except S. typhimurium. A study conducted with alkaloid extract from bark of Holarrhena pubescens showed highest bactericidal activity against S. aureus, followed by P. aeruginosa, E. coli and B. subtilis (Chakraborty and Brantner, 1999). The MIC ranged between 31-500 and is found to be in accordance with the antibacterial activity and differential distribution of bioactive alkaloid noticed in Aconitum species. Chakraborty and Brantner, (1999) reported the MIC value of alkaloid extract from bark of Holarrhena pubescens as 95, 600, 550 and 420 µg/ml for S. aureus, B. subtilis, E. coli, and P. aeruginosa, respectively and these values are quite close to our observation.

Conclusion
From the present study we conclude that in the spring, vigorous growth takes place and the parent tuber sends out (the future flowering) stem and leave. At the same time, the daughter tuber starts to develop, and it continues to grow throughout the summer and autumn. Towards the