# Phytochemical Evaluation, Antimicrobial Activity, and Determination of Bioactive Components from Leaves ofAegle marmelos

Authors: Farina Mujeeb; Preeti Bajpai; Neelam Pathak
Journal: BioMed Research International
 (2014)

*
63.9
±
0.061
*
*
29.4
±
0.004
*
*
11.98 ± 0.20
8.32 ± 0.40
NB-9
10.7 ± 0.15
19.8 ± 0.058
10.22 ± 0.032
5.36 ± 0.15
3.78 ± 0.35
NB-5
9.56 ± 0.10
23.2 ± 0.055
12.6 ± 0.005
6.57 ± 0.25
6.43 ± 0.05
AM-4
11.6 ± 0.14
23.9 ± 0.053
11.4 ± 0.007
4.23 ± 0.44
4.97 ± 0.10
NB-7
6.56 ± 0.25
18.5 ± 0.051
10.0 ± 0.005
4.65 ± 0.10
3.45 ± 0.25
AM-7
4.28 ± 0.30
39.1 ± 0.049
19.6 ± 0.045
13.40
±
0.30
5.76 ± 0.35
AM-3
14.34 ± 0.20
28.9 ± 0.048
17.78 ± 0.079
12.76 ± 0.20
7.24 ± 0.15
NB-1
3.78 ± 0.15
10.4 ± 0.047
5.8 ± 0.085
4.21 ± 0.45
3.20 ± 0.25
Kaghzi
5.94 ± 0.15
17.4 ± 0.046
11.4 ± 0.004
5.85 ± 0.35
5.31 ± 0.18
NB-4
10.57 ± 0.30
12.4 ± 0.043
9.6 ± 0.004
7.50 ± 0.24
3.60 ± 0.42
NB-16
5.80 ± 0.25
10.4 ± 0.007
7.6 ± 0.007
3.20 ± 0.15
4.35 ± 0.36
P.Sujana
7.45 ± 0.45
16.4 ± 0.070
15.82 ± 0.007
6.02 ± 0.30
6.62 ± 0.28
P.Sujata
11.98 ± 0.25
23.9 ± 0.088
13.64 ± 0.008
8.53 ± 0.25
6.43 ± 0.08
AM-1
12.79 ± 0.20
23.5 ± 0.225
15.22 ± 0.082
7.89 ± 0.55
4.05 ± 0.24
AM-2
8.43 ± 0.35
12.4 ± 0.045
8.6 ± 0.084
5.9 ± 0.34
5.32 ± 0.31
AM-6
15.03 ± 0.40
26.9 ± 0.108
20.22 ± 0.010
10.94 ± 0.28
10.54
±
0.05
AM-8
13.36 ± 0.25
26.6 ± 0.078
18.38 ± 0.078
9.25 ± 0.55
7.29 ± 0.18
**refers to significant values (P
<
0.01).


## 3.2. Antibacterial Study

The pharmacological action of the plant cannot be ascertained by the result of phytochemical studies only. Thus the antibacterial activity against pathogenic bacteria was also evaluated. The present investigation shows the efficacy of all the extracts against the selected pathogenic bacteria (Tables3, 4, and 5).Table 3
Zone of Inhibition by aqueous extract.
Aqueous extract
Conc. (mg/mL)
10
15
20
25
30
40
Standard
Control
Name of  bacteria
Zone of inhibition (mm)
Ampicillin (10 mcg/disc)
DMSO
K. pneumoniae
7.33 ± 0.03
8.33 ± 0.03
8.5 ± 0.02
6.5 ± 0.02
9.83 ± 0.04
10.6 ± 0.03
21.3 ± 0.088
0
B. cereus
7.33 ± 0.03
8.33 ± 0.03
9.00
9.00
9.00 ± 0.05
9.33 ± 0.05**
10.3 ± 0.088
0
E. aerogenes
8.00
8.00
8.33 ± 0.03
9.00
9.00
9.00**
11.3 ± 0.088
0
S. aureus
8.66 ± 0.03
9.66 ± 0.03
10.6 ± 0.03
11.6 ± 0.03
12.6 ± 0.03
13.0
40.6 ± 0.120
0
S. epidermidis
9.66 ± 0.03
11.0
12.0
13.0
13.6 ± 0.03
14.3 ± 0.06*
20.6 ± 0.145
0
Values are expressed as mean± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennett’s t test; P
*
<
0.05; P
*
*
<
0.01.Table 4
Zone of inhibition by methanol extract.
Methanol extract
Conc. (mg/mL)
10
15
20
25
30
40
Standard
Control
Name of  bacteria
Zone of inhibition (mm)
Ampicillin (10 mcg/disc)
DMSO
K. pneumoniae
9.3 ± 0.03
9.6 ± 0.03
10.6 ± 0.03
11.3 ± 0.03
11.0 ± 0.05
11.0 ± 0.05*
21.3 ± 0.088
0
B. cereus
8.6 ± 0.03
10.0
10.6 ± 0.03
11.3 ± 0.03
10.6 ± 0.03
12.0 ± 0.05**
10.3 ± 0.088
0
E. aerogenes
—
—
—
—
—
—
11.3 ± 0.088
0
S. aureus
10.0
11.0
11.0
12.3 ± 0.03
12.0
13.0
40.6 ± 0.120
0
S. epidermidis
8.0 ± 0.05
9.3 ± 0.06
10.0
10.3 ± 0.03
11.0
12.0*
20.6 ± 0.145
0
Values are expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennett’st test; P
*
<
0.05; P
*
*
<
0.01.Table 5
Zone of inhibition by aqueous: ethanol extract.
Aqueous: ethanol
Conc. (mg/mL)
40
80
120
160
Name of  bacteria
Zone of inhibition (mm)
K. pneumonia
9.0 ± 0.05
11.0 ± 0.05
12.3 ± 0.03
14.6 ± 0.06*
B. cereus
9.0
10.0
11.0
11.3 ± 0.03
E. aerogenes
10.0
15.0
16.0
18.3 ± 0.03**
S. aureus
10.3 ± 0.03
11.6 ± 0.03
12.0
13.0
S. epidermidis
10.3 ± 0.03
12.3 ± 0.03
13.3 ± 0.06
14.6 ± 0.03*
Values are expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dennett’st test; P
*
<
0.05; P
*
*
<
0.01. The standard antibiotic and control are the same as that of aqueous and methanolic extract.The aqueous extract showed highest antibacterial activity againstS. epidermidis (14.3 mm) followed byS. aureus (13 mm) andK. pneumonia(10.6 mm).The maximum zone of inhibition was observed at the concentration of 40 mg/mL. Varying degree of antibacterial activity by leaf extracts ofAegle marmelos against various tested bacterial species has been reported [37]. The presence of the compounds Cuminaldehyde and Eugenol may be responsible for the antibacterial activity of leaf extracts against various bacterial strains [38]. The aqueous, acetone, and petroleum ether extracts ofA. marmelos were found to be effective againstB. coagulans,B. subtilis,B. thuringiensis,P. aeruginosa, andS. aureus as reported earlier [39]. The dried fruit extract ofAegle marmelos showed potential antibacterial activity againstP. aeruginosa,S. flexneri,E. coli,B. subtilis,S. epidermidis, andS. aureus [40].On the other hand, the methanol extract exhibited a much better antibacterial activity as compared to aqueous extract againstK. pneumoniae,B. cereus, andS. aureus. The methanol extract was most potent againstS. aureus, showing the maximum zone of inhibition at the concentration of 40 mg/mL. The highest sensitivity ofS. aureus may be due to its cell wall structure and outer membrane [41]. No activity was seen againstE. aerogenes. A few reports have also mentioned significantin vitro antimicrobial activity by the methanol extracts ofAegle marmelos leaves and flowers [35, 42]. The MIC value of both aqueous and methanol extract was significant at 10 mg/mL; however, for aqueous: ethanol it was 40 mg/mL which is analogous to the MIC values reported using crude extracts of other medicinal plants. The antimicrobial activity of papaya seed extract with MIC value of 11.8 mg/mL was found to be effective againstS. typhiby Yismaw et al. [43]. InZizyphussp. MIC values of 25 mg/mL [44] have been reported againstS. aureus. Similarly, in case ofF. religiosa,the final MIC values were 2–4-fold higher than initial MIC which ranged from 4.7 to 18.8 mg/mL and from 2.4 to 18.8 mg/mL for resistant and for sensitive isolates, respectively. MIC values (25 mg/mL) were reported by Valsaraj et al. [44] while higher concentration of 150 mg/mL was reported by Ahmad and Beg [45] againstS. aureus.In yet another study, the antibacterial activity of whole plant ofBorreria hispida(Linn) was evaluated against various organisms, where the MIC of methanolic extract ofBorreria hispidawas found to range from 250 μg/mL to 50 mg/mL. Moreover higher MIC values ranging from 0.125 to 32 mg/mL were also reported against at least one of the test microorganisms from nine ethnobotanically Indian medicinal plants [46].The high antibacterial activity in the methanolic extract may be due to the presence of tannins, flavonoids, and terpenoids. These medicinally bioactive components exert antimicrobial action through different mechanism. Tannins cause inhibition in the cell wall synthesis by forming irreversible complexes with prolene rich protein [47]. The saponins have the ability to cause leakage of proteins and certain enzymes from the cell [48]. Terpenoids are responsible for dissolution of the cell wall of microorganism by weakening the membranous tissue [49]. Flavonoids which have been found to be effective antimicrobial substances against a wide array of microorganismsin vitro are known to be synthesized in response to microbial infection by plants. They have the ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls [50]. Furthermore, steroids are known for their antibacterial activity specifically associated with membrane lipids and cause leakage from liposomes [51].In the case of aqueous: ethanol, the maximum antibacterial activity was seen againstE. aerogenes followed byS. epidermidis andK. pneumonia(Table 5). At the highest concentration of 160 mg/mL it showed 18.3 mm zone of inhibition. The antibiotic susceptibility showed that among all the bacterial strainsS. aureus was found to be more susceptible to ampicillin followed byK. pneumonia.


## 3.3. GC-MS Analysis

In all, thirty-three compounds were identified from the GC-MS analysis of methanolic extract of Bael leaves exhibiting various phytochemical activities and were predominantly responsible for the antimicrobial activity found in the extract against the pathogenic bacteria. The retention time and percentage peak of various bioactive compounds are presented in Table6. The major phytoconstituents present in the leaf extract were 1-Dodecanol (4.83), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-(1.11), 2,3Dioxabicyclo[2.2.2]oct-5-ene, 1-Methyl-4-(1-Methylethyl)-(Limonene dioxide 1) occupying two peak areas, that is, (0.53) and (0.40), Bicyclo[3.1.1] heptane-2,3-diol, 2,6,6-trimethyl (2,3-Pinanediol)(0.97), 2-Cyclohexen-1-one, 4-hydroxy-3-methyl-6-(1-methylethyl)-(0.63, 0.41, 0.17), Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-(BHT) (0.87), Tetradecanoic acid (Myristic acid) (2.00), 2(4H)-Benzofuranone 5,6,7,7A-Tetrahydro-6-hydroxy-4,4,7a-trimethyl,1,3-cyclohexadiene,2-methyl-5-(1methylethyl)-(1-Phellandrene), 2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl ester (Cinnamic acid, 4-hydroxy-3-methoxy-, methyl ester), 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, commonly known as phytol; a diterpene has significant antimicrobial properties against many bacterial strains [52]. 9,12,15-Octadecatrienoic acid, methyl ester (Linolenic acid, methyl ester) showing antibacterial and anticandidal activity, 2-Hexadecen-1-ol, 3,7,11,15-Tetramethyl (Phytol isomer) (6.37), Octadecanoic acid (Stearic acid) (4.07), Benzene, 1,2-dimethoxy-4-[[(4 methylphenyl) sulfonyl]methyl (10.76), fatty alcohols such as Ergost-5-en-3-ol, (3.beta.) (campesterol), Stigmasta-5, 22-dien-3-ol, Stigmast-5-en-3-ol, (3.beta.)- may be synergistically responsible for the antimicrobial activity.Table 6
Activity of the phytocomponents identified from methanolic leaf extract ofAegle marmelos.
S. no.
R.T
Compound name
Molecular formula
MW
Peak area %
Compound nature
**Activity
1
7.178
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
C6H8O4
144
1.11
Flavonoid fraction
Antimicrobial, anti-inflammatory, antiproliferative
2
6.555
1-Butanol, 3-methyl-, acetate
C7H14O2
130
5.32
Alcoholic compound
Antimicrobial
3
12.09
2,3 Dioxabicyclo[2.2.2]oct-5-ene, 1-methyl-4-(1-methylethyl)-(Limonene dioxide 1)
C10H16O2
168
0.53
Terpene
antimicrobialactivity
4
13.385
Bicyclo[3.1.1]heptane-2,3-diol, 2,6,6-trimethyl(2,3-Pinanediol)
C10H18O2
170
0.97
Terpene
antimicrobialactivity
4
13.762
2-Cyclohexen-1-one, 4-hydroxy-3-methyl-6-(1-methylethyl)-
C10H16O2
168
0.63
antibacterial
5
15.203
1-Dodecanol
C12H26O
186
4.83
long-chain fatty alcohol
antibacterial
6
16.143
Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl(BHT)
C15H24O
220
0.87
Antimicrobial, antioxidantactivity
7
16.457
Benzoic acid, 4-ethoxy-, ethyl ester
C11H14O3
194
0.33
Aromaticacid ester
Antimicrobial Preservative
8
18.101
2-Propanol, 1,1′-[(1-methyl-1,2-ethanediyl)bis(oxy)]bis-(Tripropylene glycol)
C9H20O4
192
0.87
Antimicrobialactivity
9
20.189
1-Tetradecanol, acrylate
C17H32O2
268
1.46
Fatty acid esters
Anti-inflammatory,antimicrobial
10
20.523
1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid(Quinic acid)
C7H12O6
192
0.39
Aromatic acid
antimicrobialactivity, anti-inflammatory
11
21.839
Tetradecanoic acid(Myristic acid)
C14H28O2
228
2.00
Fatty acid
Antifungal, Antioxidant, cancer preventive,nematicide, hypercholesterolemic,Lubricant
12
22.070
2(4H)-Benzofuranone 5,6,7,7a-tetrahydro-6-hydroxy-4,4,7a-trimethyl
C11H16O3
196
0.26
Triterpene
antimicrobial
13
22.262
1-Heptadecanol(1-Eicosanol)
C17H36O
256
0.33
Aliphatic alcohol
Antimalarial, antifungal, Antioxidant
14
22.409
1,3-Cyclohexadiene, 2-methyl-5-(1-methylethyl)-(1-Phellandrene)
C10H16
136
0.23
Monoterpene
Antibacterial
15
22.736
1,6-Octadiene, 7-methyl-3-methylene(beta.-myrcene)
C10H16
136
0.13
Monoterpene
Antibacterial
16
23.404
2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl ester(Cinnamic acid, 4-hydroxy-3-methoxy-, methyl ester )
C11H12O4
208
0.66
Aromatic methyl esters
Antimicrobial,antioxidant, antiviral
17
23.819
Pentadecanoic acid
C15H30O2
242
0.08
Fatty acid
Antibacterial
18
24.897
3,7,11,15-Tetramethyl-2-hexadecen-1-ol(Phytol)
C20H40O
296
0.32
Diterpene
Antimicrobial, anticancer, anti-inflammatory, anti-diuretic,
19
24.986
hexadecanoic acid, methyl ester(Palmitic acid methyl ester)
C17H34O2
270
0.72
Fatty acid methyl ester
Antioxidant, hypocholesterolemic nematicide, pesticide, antiandrogenic flavor, hemolytic, 5-Alpha reductase inhibitor
20
24.951
Pentadecanoic acid
C15H30O2
242
7.99
Saturated fatty acid
Antimicrobial
21
27.266
9-Octadecenoic acid
C18H34O2
282
0.31
Unsaturated fatty acid
antibacterial
22
27.664
Heptadecanoic acid
C17H34O2
270
0.39
Saturated fatty acid
Antimicrobial
23
28.298
9,12,15-Octadecatrienoic acid, methyl ester(Linolenic acid, methyl ester)
C19H32O2
292
1.07
Fatty acid methyl ester
Antibacterial and anticandidalAntiinflammatory, hypocholesterolemic, cancer preventive, hepatoprotective, nematicide, insectifuge antihistaminic, antiarthritic, anticoronary, antieczemic antiacne, 5-Alpha reductase inhibitor Antiandrogenic
24
28.554
2-Hexadecen-1-ol, 3,7,11,15-tetramethyl(Phytol isomer)
C20H40O
296
6.37
Diterpene
AntimicrobialAnti-inflammatory Anticancer Diuretic
25
29.227
Cis-9-Hexadecenal
C16H30O
238
11.40
Aldehyde
Antimicrobial
26
29.573
Octadecanoic acid(Stearic acid)
C18H36O2
284
4.06
Fatty acid
Antimicrobial
27
41.240
Benzene, 1,2-dimethoxy-4-[
[(4 methylphenyl)sulfonyl]methyl
C16H18O4S
306
10.76
Aromatic sulfur compound
Antimicrobial
28
44.155
Cholest-5-en-3-ol (3.beta.)-
C27H46O
386
0.24
Steroidal
Antibacterial
29
45.238
Ergost-5-en-3-ol, (3.beta.)-
C27H46O
386
0.50
Steroidal
Antimicrobial,anti-inflammatoryeffects
30
45.509
Stigmasta-5,22-dien-3-ol
C29H48O
412
0.63
Steroidal
Antioxidant,antibacterial activity, antiinflammatory, antiarthritic antiasthma, diuretic
31
46.038
Stigmast-5-en-3-ol, (3.beta.)-
C29H50O
414
3.08
Steroidal
Antimicrobial antioxidant antiinflammatory antiarthritic antiasthma diuretic
32
47.033
Vitamin E
C29H50O2
430
0.58
Antioxidant and Antimicrobial activityAnalgesic, Antidiabatic Antiinflammatory, Antidermatitic, Antileukemic, Antitumor, Anticancer, Hepatoprotective, Antispasmodic
33
44.454
alpha.-Tocopherol
C29H50O2
430
0.26
Anti-inflammatory, antioxidant, antimicrobial,radical scavenging, antispasmodic
*
*Source: Dr. Duke’s: Phytochemical and Ethnobotanical Databases.The different phyto compounds responsible for bioactivity have been identified and characterized in different medicinal plants. 4H-Pyran-4-one, 2,3-dihydro-3, 5-dihydroxy-6-methyl-, a potent anti-inflammatory and antioxidant compound, possessed antibacterial activity inBarleria prionitis (Linn.) rhizome. A long-chain fatty alcohol, 1-Dodecanol, was reported with highest antibacterial activity againstStaphylococcus aureus [53]. Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl commonly known as Butylated hydroxytoluene (BHT), an antioxidant, has also demonstrated marked antimicrobial activity inhibiting or decreasing the growth of gram-positive bacteria at a higher degree than the gram-negative bacteria belonging to the family Enterobacteriaceae [54]. Derivatives of cinnamic acid such as esters, amides, acids, and hydrazides have been reported to have antibacterial, antifungal, and antiviral properties [55]. 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl, a bioactive compound possessing the properties such as analgesic, antidiabetic, antibacterial, and antifungal, was identified from the methanolic fractions of theAzadirachta indica [56]. The extract contained (0.23%) of 1,3-Cyclohexadiene, 2-methyl-5-(1-methylethyl), commonly known as 1-phellandrene. Stigmasterol has been reported earlier as a strong antioxidant having antibacterial activity against multidrug resistant mycobacteria [57, 58].Several bioactive compounds, namely, Marmin [59] and Marmelosin coumarin derivatives [60] and Aegeline, an alkaloid, have been previously reported fromAegle marmelos [61]. However the flavonoids and phenolics present in significant amount in this plant are still unexplored. The preferential quantity of these compounds in the methanolic extract of Pant Aparna as revealed by the present study directed to focus towards the purification and characterization of potential compound from this variety.


## 4. Conclusion

Medicines derived from plants have made immense contribution towards the betterment of human health and act as a source of inspiration for novel drug compounds. From the above research it can be concluded that this plant has immense potential to be used in the area of pharmacology and as a prospective source of valuable drugs. Due to the presence of various compounds that are essential for good health, it can also be used to improve the health status of society. The extracts showed a significantly high antibacterial activity against the microorganisms. The data clearly depicts the presence of compounds used for treating various bacterial diseases, indicating its use in the traditional system of medicine since ancient times. Further, the broad spectrum activity of aqueous, methanol, and aqueous: ethanol extracts proves to be encouraging in the development of novel antimicrobial formulation in the near future.A spectrum of compounds showing strong antibacterial, antioxidant, and anti-inflammatory activities was revealed by the GC-MS analysis of the methanolic extract ofAegle marmelos.Antimicrobials derived from plants possess vast curative properties since they have fewer side effects as compared to synthetic antimicrobials drugs.Aegle marmelos is of utmost importance for ethnobotanical purposes, and it has been placed in the priority list of thirty-two medicinal plants by The National Medicinal Plants Board of Govt. of India [62]. The present study contributes to the current knowledge of presence of various phytochemical active compounds in 18 varieties/accessions ofAegle marmelos possessing significant broad spectrum antibacterial efficacy. Further fractionation and purification will elucidate the potential compound, which is a pressing need because of the upcoming resistance of the currently available antibiotics.



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*Source: 497606-2014-05-11.xml*