# Activity ofAristolochia bracteolata against Moraxella catarrhalis

Authors: Malik Suliman Mohamed; Mona Timan Idriss; Amgad I. M. Khedr; Haidar Abd AlGadir; Satoshi Takeshita; Mohammad Monir Shah; Yoshio Ichinose; Toshihide Maki
Journal: International Journal of Bacteriology
 (2014)

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## Abstract

A bioassay-guided fractionation of methanol extract ofAristolochia bracteolata whole plant was carried out in order to evaluate its antimicrobial activity and to identify the active compounds in this extract. Antibacterial and antifungal activities of methanol extract against gram-positive, gram-negative, and fungal strains were investigated by the agar disk diffusion method. Among the strains tested, Moraxella catarrhalis and sea urchin-derived Bacillus sp. showed the highest sensitivity towards the methanol extract and hence they are used as test organisms for the bioassay-guided fractionation. From this extract, aristolochic acid 1 (AA-1) has been isolated and has showed the greatest antibacterial activity against both standard strain and clinical isolates of Moraxella catarrhalis with equal minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 25 and 50 μg/mL. Modification of the AA-1 to AA-1 methyl ester completely abolished the antibacterial activity of the compound and the piperonylic acid moiety of AA-1 which suggested that the coexistence of phenanthrene ring and free carboxylic acid is essential for AA-1 antibacterial activity.


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## Body

## 1. Introduction

Moraxella catarrhalis is a gram-negative, aerobic diplococcus human mucosal pathogen which causes middle ear infections in infants and children [1–3], and it is one of the three major causes of otitis media along withStreptococcus pneumoniaandHaemophilus influenzae[4]. AlthoughMoraxella catarrhalisis frequently found as a commensal of the upper respiratory tract, recently it has emerged as a genuine pathogen and is now considered an important cause of upper respiratory tract infections in healthy children and elderly people, lower respiratory tract infections in adults with chronic obstructive pulmonary disease [1, 5], and hospital-acquired pneumonia [6]. Amikacin, cefixime, fosfomycin, cefuroxime, cotrimoxazole, doxycycline, and erythromycin resistant strains ofMoraxella catarrhalis were isolated and the widespread production of a β-1actamase enzyme renders the bacterium resistant to the penicillin [7–9].This has led to the search for new and effective therapeutic alternatives among natural compounds. Plants remain an important source of diverse chemical entities which have been used as drugs or provide scaffolds from which new drugs have been derived [10]. The selection of a suitable candidate species for investigations can be done on the basis of long-term use by humans. This approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use [11].Aristolochia is an important genus in the family ofAristolochiaceae and is widespread across tropical Asia, Africa, and South America.Aristolochia bracteolata is commonly called “worm killer” in English due to supposed anthelmintic activity and trypanocidal effect [12]. It is used in traditional medicine as a gastric stimulant and in the treatment of cancer, lung inflammation, dysentery, and snakebites [13].Aristolochiaceae has been used by Sudanese people as analgesic, antiscorpion, and antisnake. It is also used in the treatment of tumors and malaria and for fevers [14], but its usage as an antimalarial is not recommended in its crude form.Aristolochia bracteolata showed a definite positive effect on wound healing, with significant increase in the level of powerful antioxidant enzymes. Its root and leaves were bitter and anthelmintic and are medicinally important. Almost every part of the plant has medicinal usage [15]. The whole plant was used as a purgative, antipyretic, and anti-inflammatory. It also possesses a potent antiallergic activity [16]. Organic solvent extracts of the plant showed antibacterial activities while the water extract showed antifungal activity [17]. The plant also showed promising antiarthritis activity [18]. AlthoughAristolochia has been used for thousands of years in many cultures for many indications due to its various pharmacological activities, it was later discovered that consuming these plants can certainly be dangerous. The genus ofAristolochia contains a naturally carcinogenic compound AA which has been shown to be the cause of so-called Chinese herb nephropathy or AA nephropathy [19, 20], and mutations in the cells of people who consume it, causes more mutations than two of the best-known environmental carcinogens: tobacco smoke and UV light [21, 22]. There are many cases of nephropathy reported in the literature caused by the systemic and long term application of Chinese snakeroot (Aristolochia fangchi); this highlighted the risk of using preparations which contain aristolochic acids [23].AlthoughAristolochia is being known in many countries that is containing a toxic compound AA, but this has not stopped it from being a popular herbal remedy for thousands of years. It is still extensively used in India and in traditional Chinese medicine for slimming, menstrual symptoms, and rheumatism. It is also widespread used in Sudan and other African countries as one of the most effective herbal remedies for infectious diseases. Therefore, it was our objective to assess the potential antimicrobial activity ofAristolochia bracteolata using a bioassay-guided fractionation, in order to produce pure compound that can act as the lead compound in developing new, safe, and effective drug to replace the use of the harmful crude plant material.


## 2. Materials and Methods

### 2.1. Materials

#### 2.1.1. General

Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F
254
s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High resolution FAB-MS and ESI-MS were recorded on JEOL JMS700N and JMS-100TD, respectively. 1H- and 13C-NMR, 1H-1H COSY, NOESY, HSQC, and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian Inc., U.S.A.) operating at 500 MHz for 1H and 125 MHz for 13C, respectively. 1H-NMR chemicals shifts are expressed in δ values referring to the solvent peak δ
H 2.49 for DMSO and coupling constants are expressed in Hz. 13C-NMR chemical shifts are expressed in δ values referring to the solvent peak δ
C 39.5 for DMSO. Piperonylic acid was purchased from commercial sources (TCI) and used without further purification.


#### 2.1.2. Plant Material

The plant material (whole) was collected in the period from (October to December 2012) from Khartoum state in Sudan. The plant was kindly identified and authenticated by the Taxonomist Dr. Haider Abdelgadir and Mr. Yahia Mohammed, Medicinal and Aromatic Plants Research Institute (MAPRI).


#### 2.1.3. Test Microorganisms

(a)
Standard strains:Moraxella catarrhalis (GTC 01544),Klebsiella pneumoniae (ATCC 13883),Escherichia coli (K12),Salmonella typhimurium (ATCC 14028),Streptococcus pyogenes (ATCC 19615),Streptococcus agalactiae (ATCC 13813),Staphylococcus epidermidis(ATCC 12228),Neisseria lactamicus (ATCC 23970),Enterobacter cloacae, (ATCC 23355),Bacillus subtilis (ATCC 6633),Staphylococcus aureus (209P), andPseudomonas aeruginosa (IFO 3445).
(b)
Clinical strains: Moraxella catarrhalis, Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae,andYersinia enterocolitica.In addition to a sea urchin (Anthocidaris crassispina) derivedBacillus sp. which obtained from the Laboratory in Medical Plants Garden, Nagasaki University.(c)
Fungal strains: the fungal strains used wereAspergillus niger (NBRC 33023),Penicillium crustosum (NBRC 33015),Schizophyllum commune (NBRC 30749),Trichophyton concentricum (NBRC 31068), andCandida albicans (NBRC 10108).


### 2.2. Extraction of Plant Material

The air-dried powdered whole plant (200 g) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature. The methanolic extract was passed through Whatman number 1 filter paper (Whatman England) and the concentrated extract (40 g) was digested with 100 mL distilled water and successively partitioned withn-hexane (4 × 400 mL), chloroform (3 × 400 mL), ethyl acetate (5 × 400 mL), andn-butanol (2 × 400 mL). Each fraction was concentrated under reduced pressure to a constant weight to give the correspondingn-hexane fraction (0.4 g), chloroform fraction (2 g), ethyl acetate fraction (0.7 g),n-butanol fraction (6 g), and aqueous fraction (30 g).The most active fraction againstBacillus sp. andM. catarrhalis (chloroform fraction) was subjected to Sephadex LH20 column chromatography to give three subfractions (A-C). Fraction (B) was resubjected again to Sephadex LH20 to afford very active and pure compound AA-1 (150 mg).


### 2.3. Preparation of Methyl Ester of AA-1

To the solution of AA-1 (50 mg; 0.23 mmol) in dimethylformamide (DMF), 1 mL potassium carbonate was added (95 mg; 0.69 mmol). To the resulting suspension iodomethane was added (72μL; 1.15 mmol) and stirred for 8 hours. The reaction mixture was poured onto water (10 mL) and ethyl acetate (20 mL). The organic layer was washed with 1 M HCl (10 mL) three times and then with Brine (10 mL) once. The resulting solution was dried over magnesium sulphate and filtrated. After removal of solvent under reduced pressure, the residue was purified by silica-gel chromatography (chloroform-methanol) to afford the titled ester (88%). 1H-NMR (400 MHz, CDCl3, TMS, r.t.) δ (ppm): 3.88 (3H, s), 4.06 (3Hs), 6.38 (2H, s), 7.11 (1H, d,J = 7.8 Hz), 7.72 (1H, dd,J = 7.8 Hz, 8.0 Hz), 7.77 (1H, s), 8.70 (1H, d,J = 8.0 Hz), and 8.83 (1H, s).


### 2.4. Antimicrobial Activity

#### 2.4.1. Antibacterial Assay

The antibacterial activity was tested by agar disc diffusion assay [24]. Suspension of the tested bacteria (100 μL of 108 cfu/mL) was spread onto solid media plates. The sterile paper discs (6 mm in diameter) which were impregnated with the plant extract (1–4 mg) and pure compound (10–100 μg) were placed aseptically over the bacterial culture on nutrient agar plates. After incubation at 37°C for 24 hours, the zone of inhibition around the discs was measured by millimeter scale. The experiment was replicated two times to confirm the reproducible results. Sterile, blank paper discs impregnated with only sterile solvents served as negative control each time.


#### 2.4.2. Antifungal Assay

The antifungal activity was determined by disk diffusion method [25, 26]. Fungi strains were inoculated on nutrient agar plates supplemented with 2% glucose. The sterile paper discs (6 mm in diameter) which were impregnated with individual extract were placed on the inoculated plates. These plates were incubated for 24–72 h at 25–28°C and the growth was evaluated visually by comparing a particular plate with the negative control plates (having only test fungi). The antifungal activity was evaluated by measuring the inhibition zone diameter (in millimeter) observed.


#### 2.4.3. Determination of MIC and MBC

The MIC and MBC values were determined by broth dilution method in accordance with CLSI methodology [27]. Bacterial strains were cultured for 24 h at 37°C on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5 ± 1.0 × 103 cfu/mL. Dilutions ranging from 1.56 to 400 μg/mL of the compound were prepared in tubes including broth and DMSO 10% (v/v), in addition to one negative control (broth + DMSO 10% v/v + test microorganism) to ensure that the final concentration of DMSO in the assays did not interfere with the bacterial growth and one sterility control (broth + DMSO 10% v/v + test compound). A 100 μL suspension of test microorganism was added to individual tubes and incubated at 37°C for 24 h. The MIC of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the MBC was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic-free plates. Afterward results were confirmed by two trained laboratory personnel.


## 2.1. Materials

### 2.1.1. General

Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F
254
s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High resolution FAB-MS and ESI-MS were recorded on JEOL JMS700N and JMS-100TD, respectively. 1H- and 13C-NMR, 1H-1H COSY, NOESY, HSQC, and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian Inc., U.S.A.) operating at 500 MHz for 1H and 125 MHz for 13C, respectively. 1H-NMR chemicals shifts are expressed in δ values referring to the solvent peak δ
H 2.49 for DMSO and coupling constants are expressed in Hz. 13C-NMR chemical shifts are expressed in δ values referring to the solvent peak δ
C 39.5 for DMSO. Piperonylic acid was purchased from commercial sources (TCI) and used without further purification.


### 2.1.2. Plant Material

The plant material (whole) was collected in the period from (October to December 2012) from Khartoum state in Sudan. The plant was kindly identified and authenticated by the Taxonomist Dr. Haider Abdelgadir and Mr. Yahia Mohammed, Medicinal and Aromatic Plants Research Institute (MAPRI).


### 2.1.3. Test Microorganisms

(a)
Standard strains:Moraxella catarrhalis (GTC 01544),Klebsiella pneumoniae (ATCC 13883),Escherichia coli (K12),Salmonella typhimurium (ATCC 14028),Streptococcus pyogenes (ATCC 19615),Streptococcus agalactiae (ATCC 13813),Staphylococcus epidermidis(ATCC 12228),Neisseria lactamicus (ATCC 23970),Enterobacter cloacae, (ATCC 23355),Bacillus subtilis (ATCC 6633),Staphylococcus aureus (209P), andPseudomonas aeruginosa (IFO 3445).
(b)
Clinical strains: Moraxella catarrhalis, Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae,andYersinia enterocolitica.In addition to a sea urchin (Anthocidaris crassispina) derivedBacillus sp. which obtained from the Laboratory in Medical Plants Garden, Nagasaki University.(c)
Fungal strains: the fungal strains used wereAspergillus niger (NBRC 33023),Penicillium crustosum (NBRC 33015),Schizophyllum commune (NBRC 30749),Trichophyton concentricum (NBRC 31068), andCandida albicans (NBRC 10108).


## 2.1.1. General

Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F
254
s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High resolution FAB-MS and ESI-MS were recorded on JEOL JMS700N and JMS-100TD, respectively. 1H- and 13C-NMR, 1H-1H COSY, NOESY, HSQC, and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian Inc., U.S.A.) operating at 500 MHz for 1H and 125 MHz for 13C, respectively. 1H-NMR chemicals shifts are expressed in δ values referring to the solvent peak δ
H 2.49 for DMSO and coupling constants are expressed in Hz. 13C-NMR chemical shifts are expressed in δ values referring to the solvent peak δ
C 39.5 for DMSO. Piperonylic acid was purchased from commercial sources (TCI) and used without further purification.


## 2.1.2. Plant Material

The plant material (whole) was collected in the period from (October to December 2012) from Khartoum state in Sudan. The plant was kindly identified and authenticated by the Taxonomist Dr. Haider Abdelgadir and Mr. Yahia Mohammed, Medicinal and Aromatic Plants Research Institute (MAPRI).


## 2.1.3. Test Microorganisms

(a)
Standard strains:Moraxella catarrhalis (GTC 01544),Klebsiella pneumoniae (ATCC 13883),Escherichia coli (K12),Salmonella typhimurium (ATCC 14028),Streptococcus pyogenes (ATCC 19615),Streptococcus agalactiae (ATCC 13813),Staphylococcus epidermidis(ATCC 12228),Neisseria lactamicus (ATCC 23970),Enterobacter cloacae, (ATCC 23355),Bacillus subtilis (ATCC 6633),Staphylococcus aureus (209P), andPseudomonas aeruginosa (IFO 3445).
(b)
Clinical strains: Moraxella catarrhalis, Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae,andYersinia enterocolitica.In addition to a sea urchin (Anthocidaris crassispina) derivedBacillus sp. which obtained from the Laboratory in Medical Plants Garden, Nagasaki University.(c)
Fungal strains: the fungal strains used wereAspergillus niger (NBRC 33023),Penicillium crustosum (NBRC 33015),Schizophyllum commune (NBRC 30749),Trichophyton concentricum (NBRC 31068), andCandida albicans (NBRC 10108).


## 2.2. Extraction of Plant Material

The air-dried powdered whole plant (200 g) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature. The methanolic extract was passed through Whatman number 1 filter paper (Whatman England) and the concentrated extract (40 g) was digested with 100 mL distilled water and successively partitioned withn-hexane (4 × 400 mL), chloroform (3 × 400 mL), ethyl acetate (5 × 400 mL), andn-butanol (2 × 400 mL). Each fraction was concentrated under reduced pressure to a constant weight to give the correspondingn-hexane fraction (0.4 g), chloroform fraction (2 g), ethyl acetate fraction (0.7 g),n-butanol fraction (6 g), and aqueous fraction (30 g).The most active fraction againstBacillus sp. andM. catarrhalis (chloroform fraction) was subjected to Sephadex LH20 column chromatography to give three subfractions (A-C). Fraction (B) was resubjected again to Sephadex LH20 to afford very active and pure compound AA-1 (150 mg).


## 2.3. Preparation of Methyl Ester of AA-1

To the solution of AA-1 (50 mg; 0.23 mmol) in dimethylformamide (DMF), 1 mL potassium carbonate was added (95 mg; 0.69 mmol). To the resulting suspension iodomethane was added (72μL; 1.15 mmol) and stirred for 8 hours. The reaction mixture was poured onto water (10 mL) and ethyl acetate (20 mL). The organic layer was washed with 1 M HCl (10 mL) three times and then with Brine (10 mL) once. The resulting solution was dried over magnesium sulphate and filtrated. After removal of solvent under reduced pressure, the residue was purified by silica-gel chromatography (chloroform-methanol) to afford the titled ester (88%). 1H-NMR (400 MHz, CDCl3, TMS, r.t.) δ (ppm): 3.88 (3H, s), 4.06 (3Hs), 6.38 (2H, s), 7.11 (1H, d,J = 7.8 Hz), 7.72 (1H, dd,J = 7.8 Hz, 8.0 Hz), 7.77 (1H, s), 8.70 (1H, d,J = 8.0 Hz), and 8.83 (1H, s).


## 2.4. Antimicrobial Activity

### 2.4.1. Antibacterial Assay

The antibacterial activity was tested by agar disc diffusion assay [24]. Suspension of the tested bacteria (100 μL of 108 cfu/mL) was spread onto solid media plates. The sterile paper discs (6 mm in diameter) which were impregnated with the plant extract (1–4 mg) and pure compound (10–100 μg) were placed aseptically over the bacterial culture on nutrient agar plates. After incubation at 37°C for 24 hours, the zone of inhibition around the discs was measured by millimeter scale. The experiment was replicated two times to confirm the reproducible results. Sterile, blank paper discs impregnated with only sterile solvents served as negative control each time.


### 2.4.2. Antifungal Assay

The antifungal activity was determined by disk diffusion method [25, 26]. Fungi strains were inoculated on nutrient agar plates supplemented with 2% glucose. The sterile paper discs (6 mm in diameter) which were impregnated with individual extract were placed on the inoculated plates. These plates were incubated for 24–72 h at 25–28°C and the growth was evaluated visually by comparing a particular plate with the negative control plates (having only test fungi). The antifungal activity was evaluated by measuring the inhibition zone diameter (in millimeter) observed.


### 2.4.3. Determination of MIC and MBC

The MIC and MBC values were determined by broth dilution method in accordance with CLSI methodology [27]. Bacterial strains were cultured for 24 h at 37°C on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5 ± 1.0 × 103 cfu/mL. Dilutions ranging from 1.56 to 400 μg/mL of the compound were prepared in tubes including broth and DMSO 10% (v/v), in addition to one negative control (broth + DMSO 10% v/v + test microorganism) to ensure that the final concentration of DMSO in the assays did not interfere with the bacterial growth and one sterility control (broth + DMSO 10% v/v + test compound). A 100 μL suspension of test microorganism was added to individual tubes and incubated at 37°C for 24 h. The MIC of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the MBC was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic-free plates. Afterward results were confirmed by two trained laboratory personnel.


## 2.4.1. Antibacterial Assay

The antibacterial activity was tested by agar disc diffusion assay [24]. Suspension of the tested bacteria (100 μL of 108 cfu/mL) was spread onto solid media plates. The sterile paper discs (6 mm in diameter) which were impregnated with the plant extract (1–4 mg) and pure compound (10–100 μg) were placed aseptically over the bacterial culture on nutrient agar plates. After incubation at 37°C for 24 hours, the zone of inhibition around the discs was measured by millimeter scale. The experiment was replicated two times to confirm the reproducible results. Sterile, blank paper discs impregnated with only sterile solvents served as negative control each time.


## 2.4.2. Antifungal Assay

The antifungal activity was determined by disk diffusion method [25, 26]. Fungi strains were inoculated on nutrient agar plates supplemented with 2% glucose. The sterile paper discs (6 mm in diameter) which were impregnated with individual extract were placed on the inoculated plates. These plates were incubated for 24–72 h at 25–28°C and the growth was evaluated visually by comparing a particular plate with the negative control plates (having only test fungi). The antifungal activity was evaluated by measuring the inhibition zone diameter (in millimeter) observed.


## 2.4.3. Determination of MIC and MBC

The MIC and MBC values were determined by broth dilution method in accordance with CLSI methodology [27]. Bacterial strains were cultured for 24 h at 37°C on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5 ± 1.0 × 103 cfu/mL. Dilutions ranging from 1.56 to 400 μg/mL of the compound were prepared in tubes including broth and DMSO 10% (v/v), in addition to one negative control (broth + DMSO 10% v/v + test microorganism) to ensure that the final concentration of DMSO in the assays did not interfere with the bacterial growth and one sterility control (broth + DMSO 10% v/v + test compound). A 100 μL suspension of test microorganism was added to individual tubes and incubated at 37°C for 24 h. The MIC of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the MBC was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic-free plates. Afterward results were confirmed by two trained laboratory personnel.


## 3. Results and Discussion

### 3.1. Antimicrobial Activity

Initial steps in new drug discovery involve identification of new chemical entities, which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation. Bioassay-guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug [11].The whole plant ofAristolochia bracteolata was extracted successively with MeOH and subjected to liquid-liquid fractionation withn-hexane, chloroform, ethyl acetate, andn-butanol. The resulting fractions were tested for antibacterial and antifungal activities. The crude extract and chloroform fraction were significantly active against sea urchin-derivedBacillussp. and both standard strain and clinical isolates ofMoraxella catarrhalis and were moderately active againstS. aureus, B. subtilis, andPs. aeruginosa. Then-hexane fraction had moderate activity againstS. aureus andB. subtilis while ethyl acetate fraction showed moderate activity againstPs. aeruginosa andB. subtilis. All fractions were active against sea urchin-derivedBacillus sp. (Table 1). The crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains:Klebsiella pneumoniae,Escherichia coli,Salmonella typhimurium,Streptococcus pyogenes,Streptococcus agalactiae,Staphylococcus epidermidis,Neisseria lactamicus,Enterobacter cloacae,Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae, and Yersinia enterocolitica.Table 1
Antibacterial activity of crude plant extract and fractions.
Diameter of zone of inhibition (mm)
Microorganism
Crude extract
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
B. subtilis
15
12
16
12
—
MarineBacillus sp.
25
18
25
20
15
S. aureus
15
12
11
—
—
Ps. aeruginosa
10
—
11
11
—
M. catarrhalis
12
—
14
9
—The chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound AA-1 (Figure1). AA-1 showed strong activity againstMoraxella catarrhalis (standard strain and clinical isolates) and sea urchin-derivedBacillus sp. (Table 2), with equal MIC and MBC values of 25 and 50 μg/mL. Both the piperonylic acid moiety of AA-1 (Figure 1) and AA-1-methyl ester showed no activity against bacteria (Table 2), which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for AA-1 antibacterial activity.Table 2
Antibacterial activity of AA-1 and its derivatives.
Diameter of zone of inhibition (mm)
Microorganism
AA-1
AA-1 methyl ester
Piperonylic acid
Ciprofloxacin
MarineBacillus sp.
12
—
—
20
M. catarrhalisT
12
—
—
20
M. catarrhalis1 CI
11
—
—
18
M. catarrhalis 2 CI
12
—
—
19
M. catarrhalis 3 CI
12
—
—
20
M. catarrhalis 4 CI
11
—
—
20
M. catarrhalis5 CI
12
—
—
19
T: type strain; CI: clinical isolate.Figure 1
Structure of AA-1, AA-1 methyl ester, and piperonylic acid.


### 3.2. Structure Elucidation of AA-1

Bioguided fractionation of methanolic extract ofAristolochia bracteolata led to isolation of AA-1 and its structure was elucidated by interpretation of its NMR and MS data and by comparison with those reported in the literature. Electrospray ionization mass spectrometry (ESIMS) showed pseudomolecular ion signal atm/z 364.03 [M + Na]+ and high resolution fast atom bombardment mass spectrometry (HR-FABMS) afforded M+1 ion signal atm/z 342.0620 which was corresponding to the molecular formula C17H12NO7 (calculated for 342.06138). 1H NMR and 3C-NMR spectra were matched with those of AA-1 which were previously reported [28]. The correlation among each NMR signal was confirmed by 2D HSQC and HMBC. Two weak signals which did not show any correlation with proton in HSQC and HMBC spectroscopy were considered as quaternary carbons at positions 5 (δ 124.3 ppm) and 6 (δ 143.5 ppm), respectively.Laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganismsin vitro. More of these compounds should be subjected to animal and human studies to determine their effectiveness in whole-organism systems, including in particular toxicity studies and an examination of their effects on beneficial normal microbiota [10].In spite of the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects, many people believe that treatments that are natural are somehow magically safe and effective.Aristolochia is used in traditional medicine for the treatment of various diseases [13, 15], including those associated with bacteria. This study showed clearly that the excellent effect ofAristolochia in treating such diseases is due to the toxic compound AA-1. Although AA-1 is highly effective in killingM. catarrhalis, it is ineffective against the other microorganisms tested. This highlights the importance ofM. catarrhalis in discovering the cellular target of AA-1 and the mechanism of AA-1 toxicity. The widespread use ofAristolochia is not sufficient to ensure that it is effective or even that it is safe. Therefore, hit-to-lead exploration is necessary to identify related compounds with low toxicity, low cost, and improved potency that can replace the use of the harmful crude plant material.It is impossible to ban the use of these remedies, especially in the rural areas in Sudan and other African countries; therefore, we strongly recommend educating the public of the risks versus the benefits ofAristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents.


## 3.1. Antimicrobial Activity

Initial steps in new drug discovery involve identification of new chemical entities, which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation. Bioassay-guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug [11].The whole plant ofAristolochia bracteolata was extracted successively with MeOH and subjected to liquid-liquid fractionation withn-hexane, chloroform, ethyl acetate, andn-butanol. The resulting fractions were tested for antibacterial and antifungal activities. The crude extract and chloroform fraction were significantly active against sea urchin-derivedBacillussp. and both standard strain and clinical isolates ofMoraxella catarrhalis and were moderately active againstS. aureus, B. subtilis, andPs. aeruginosa. Then-hexane fraction had moderate activity againstS. aureus andB. subtilis while ethyl acetate fraction showed moderate activity againstPs. aeruginosa andB. subtilis. All fractions were active against sea urchin-derivedBacillus sp. (Table 1). The crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains:Klebsiella pneumoniae,Escherichia coli,Salmonella typhimurium,Streptococcus pyogenes,Streptococcus agalactiae,Staphylococcus epidermidis,Neisseria lactamicus,Enterobacter cloacae,Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae, and Yersinia enterocolitica.Table 1
Antibacterial activity of crude plant extract and fractions.
Diameter of zone of inhibition (mm)
Microorganism
Crude extract
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
B. subtilis
15
12
16
12
—
MarineBacillus sp.
25
18
25
20
15
S. aureus
15
12
11
—
—
Ps. aeruginosa
10
—
11
11
—
M. catarrhalis
12
—
14
9
—The chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound AA-1 (Figure1). AA-1 showed strong activity againstMoraxella catarrhalis (standard strain and clinical isolates) and sea urchin-derivedBacillus sp. (Table 2), with equal MIC and MBC values of 25 and 50 μg/mL. Both the piperonylic acid moiety of AA-1 (Figure 1) and AA-1-methyl ester showed no activity against bacteria (Table 2), which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for AA-1 antibacterial activity.Table 2
Antibacterial activity of AA-1 and its derivatives.
Diameter of zone of inhibition (mm)
Microorganism
AA-1
AA-1 methyl ester
Piperonylic acid
Ciprofloxacin
MarineBacillus sp.
12
—
—
20
M. catarrhalisT
12
—
—
20
M. catarrhalis1 CI
11
—
—
18
M. catarrhalis 2 CI
12
—
—
19
M. catarrhalis 3 CI
12
—
—
20
M. catarrhalis 4 CI
11
—
—
20
M. catarrhalis5 CI
12
—
—
19
T: type strain; CI: clinical isolate.Figure 1
Structure of AA-1, AA-1 methyl ester, and piperonylic acid.


## 3.2. Structure Elucidation of AA-1

Bioguided fractionation of methanolic extract ofAristolochia bracteolata led to isolation of AA-1 and its structure was elucidated by interpretation of its NMR and MS data and by comparison with those reported in the literature. Electrospray ionization mass spectrometry (ESIMS) showed pseudomolecular ion signal atm/z 364.03 [M + Na]+ and high resolution fast atom bombardment mass spectrometry (HR-FABMS) afforded M+1 ion signal atm/z 342.0620 which was corresponding to the molecular formula C17H12NO7 (calculated for 342.06138). 1H NMR and 3C-NMR spectra were matched with those of AA-1 which were previously reported [28]. The correlation among each NMR signal was confirmed by 2D HSQC and HMBC. Two weak signals which did not show any correlation with proton in HSQC and HMBC spectroscopy were considered as quaternary carbons at positions 5 (δ 124.3 ppm) and 6 (δ 143.5 ppm), respectively.Laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganismsin vitro. More of these compounds should be subjected to animal and human studies to determine their effectiveness in whole-organism systems, including in particular toxicity studies and an examination of their effects on beneficial normal microbiota [10].In spite of the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects, many people believe that treatments that are natural are somehow magically safe and effective.Aristolochia is used in traditional medicine for the treatment of various diseases [13, 15], including those associated with bacteria. This study showed clearly that the excellent effect ofAristolochia in treating such diseases is due to the toxic compound AA-1. Although AA-1 is highly effective in killingM. catarrhalis, it is ineffective against the other microorganisms tested. This highlights the importance ofM. catarrhalis in discovering the cellular target of AA-1 and the mechanism of AA-1 toxicity. The widespread use ofAristolochia is not sufficient to ensure that it is effective or even that it is safe. Therefore, hit-to-lead exploration is necessary to identify related compounds with low toxicity, low cost, and improved potency that can replace the use of the harmful crude plant material.It is impossible to ban the use of these remedies, especially in the rural areas in Sudan and other African countries; therefore, we strongly recommend educating the public of the risks versus the benefits ofAristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents.


## 4. Conclusion

Using bioassay-guided fractionation technique, the present study directly linked the antibacterial activity ofAristolochia bracteolata to the AA-1. Although AA-1 had strong activity againstM. catarrhalis, it had a narrow spectrum of activity than expected based on the activity of the crude extract from which it was isolated or from its traditional usage. This may be the result of synergism between different compounds in the complex extracts or may be due to placebo effect.



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*Source: 481686-2014-09-28.xml*