Formulation, Characterization and Antimicrobial efficacy of Aegle marmelos Essential oil nanogel

 

Riham Omar Bakr¹*, Soumaya Saad Zaghloul¹, Reham Ibrahim Amer^2,3,

Dalia Abd Elaty Mostafa², Mahitab Helmy El Bishbishy¹

¹Pharmacognosy Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 11787, Giza, Egypt.

²Pharmaceutics Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 11787, Giza, Egypt.

³Pharmaceutics and Industrial Pharmacy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.

 

ABSTRACT:

Objective: Aegle marmelos (L.) Correa has been widely used in Indian traditional medicine and has many reported pharmacological activities. The aim of this research was to formulate solid lipid nanoparticles (SLNs) of Aegle oil (AO) that enhanced the beneficial antimicrobial activity of the oil. Methods: The chemical composition of Aegle leaf essential oil was analysed by GC-MS. Additionally, a phytochemical study of A. marmelos methanolic leaf extract was conducted using Folin-Ciocalteu colorimetric assay for determination of total phenolic content as well as ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS-MS) analyses for identification of individual components.  Six formulations of AO-loaded SLNs (AO-SLNs) were prepared by a double emulsification method. The particle size, zeta potential (ZP), polydispersibility index (PDI) and drug encapsulation efficiency (EE) of the SLNs were determined. The morphology of the SLNs was observed by transmission electron microscopy (TEM). The antimicrobial activity of AO and AO-SLNs was assessed using disc diffusion method. Results: Thirty-two compounds were identified in the Aegle oil, of which Δ-carene and α-phellandrene were the most abundant (48.14% and 34.14%, respectively). The estimated total phenolic content was 968mg gallic acid equivalents (GAE)/g, while UPLC-ESI-MS-MS led to the tentative characterization of thirteen metabolites. The SLNs showed ZP, PDI and EE 125 ± 0.22nm, –37.85, 0.282, and 92%, respectively. AO and AO-SLNs showed significant antimicrobial activity, and the SLNs could sustain the release of AO from their gel vehicles. Conclusion: Our results provide evidence for the application of AO-SLNs in topical and transdermal delivery systems.

 

 

 

INTRODUCTION:

Medicinal plants are the main sources of candidate compounds for drug discovery worldwide¹. Essential oils (EOs) are volatile, natural, aromatic oily liquids mostly of plant origin, varying in concentration between its different organs. The variety in the bioactivity of EOs has been confirmed and well correlated with their chemical composition². All the cosmetic and pharmaceutical formulations containing EOs are formulated by dispersing the EOs in semisolid media. The highly hydrophobic nature of EOs necessitates the addition of fat-like excipients for their use in topical preparations. However, EOs have many restrictions because they cannot be applied directly on the skin due to a risk of allergic reactions, being susceptible to oxidation and high volatility without a vehicle. In addition, the low solubility of EOs affects their bioavailability and the chosen route of administration. Solid lipid nanoparticles (SLNs) comprise a solid lipid core stabilized by a surfactant at the interfacial region with better physical stability, increased stability, high drug payload capacity and no carrier biotoxicity³. The lipoidal nature of these nanoparticles enables good interaction with several cell types; therefore, such particles may be helpful in the treatment of microbial infections⁴. Moreover, the entrapment of EOs enhances their chemical and biological activity, increases their stability, and enables their controlled release^5,6.

 

Aegle marmelos (L.) Correa (Rutaceae), commonly known as Bael, is widely used in Ayurvedic medicine and has also been used by many ethnic communities residing in India for over 5000 years⁷. The leaves, roots, bark, seeds and fruits are edible and of great medicinal value. This magic plant contains numerous constituents, including carotenoids, phenolics, alkaloids, pectins, tannins, coumarins, flavonoids and terpenoids^7,8. The seeds also contain anthraquinones^9,10. Pharmacological studies have reported potent anti-inflammatory and analgesic¹¹; antimicrobial^12,13; hypoglycaemic and antihyperlipidemic^14,15; antioxidant¹⁶; hepatoprotective ^17,18; and cytotoxic¹⁹ activities.

 

In this study, the leaves of A. marmelos cultivated in Egypt were subjected to a phytochemical evaluation in which the main phytoconstituents were determined.  Aegle oil (AO) was analysed and encapsulated using a double emulsification technique. Then, the optimized AO-loaded SLNs (AO-SLNs) were formulated into a hydrogel that has been evaluated and investigated for its antifungal and antimicrobial activities.

 

MATERIALS AND METHODS:

Materials:

Tween 80®, cholesterol and triethanolamine were purchased from Sigma Aldrich (St Louis, MO). Soybean lecithin (Lipoid S 100) and Compritol 888 ATO® were purchased from Lipoid GmbH (Ludwigshafen, Germany). Dimethylsulphoxide, methyl alcohol and Carbopol 934 were purchased from Merck Ltd. The other solvents used were of analytical grade.

 

Plant material:

Leaves of A. marmelos (L.) Correa were collected from El-Zohria Garden in September 2017 and identified by Dr. Therese Labib (Consultant of Taxonomy at the Ministry of Agriculture). A voucher specimen of the plant (RS-11) was kept at the Herbarium of Pharmacognosy department, Faculty of Pharmacy, MSA University.

 

Preparation of the volatile oil and aqueous methanolic extract:

Three hundred grams of fresh leaves were distilled using a Clevenger apparatus. The yield obtained was calculated, and the oil was dried over anhydrous sodium sulphate and stored at -20ºC until GC-MS analysis and microbiological investigation. Further, 300g of leaves were air dried, comminated and refluxed with 70% methanol. The collected extract was concentrated under reduced pressure on a rotary evaporator until dryness. The dried extract was retained for phytochemical and antimicrobial investigations.

 

Characterization of A. marmelos essential oil:

Determination of the essential oil constituents was performed using gaseous chromatography coupled to mass spectrometry (GC) on a Shimadzu QP2010 GC-MS system equipped with an Rtx-5MS column (30m × 0.25 mm × 0.25µm; Restek, USA) and previously adopted and reported conditions²⁰. The percentage of each oil constituent was determined by calculating the average areas under the peaks in three independent chromatographic runs and compared with a homologous series of n-alkanes (C8 – C18; Aldrich Chemical Company, USA) injected under the same conditions. The essential oil components were identified by comparing their mass spectra and calculated Kovats retention indices (KIs) with those of the available reference compounds and the data in the Adams library²¹.

 

Estimation of the total phenolic content:

The total phenolic content in the leaf extract was determined by using Folin-Ciocalteu reagent (FCR), gallic acid as a standard and measuring the maximal absorption at 765nm²². Measurements were carried out in triplicate, and calculations were based on a gallic acid calibration curve. The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry extract.

 

Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS-MS):

The leaf extract was prepared at a concentration of 40µg in 1ml methanol and injected into the UPLC/ESI-MS-MS. UPLC analysis was conducted on an Agilent 1100 series instrument using a Knauer column (250 × 2mm, ID) pre-packed with Eurospher 100-5 C18 and an integrated pre-column. The mobile phase (Acetonitrile: Nano Pure H₂O with 0.1% formic acid) with a gradient program (from 10% to 100% Acetonitrile in 35 min) was used. For mass spectrometric analysis, a Finnigan LCQ-DECA mass spectrometer with a PDA detector and a standard flow cell (10mm path length, 14µl volume, 40 bar maximum pressure) was used. The samples were detected in the negative and positive modes over a mass range of m/z 100–2000.

 

Preparation of AO-SLNs:

Six formulations of AO-SLNs were successfully prepared by a double emulsification technique (Table 1). The effects of the lipid mixture (cholesterol: lecithin) or (Compritol 888 ATO: lecithin) concentrations on the prepared AO-SLNs were investigated. The aqueous phase of a methanolic solution containing AO was poured into the organic phase; surfactant was added, which reduced the interfacial tension between the phases; and the mixture was homogenized for 15 min. Uniformly sized globules were formed as a w/o emulsion. This emulsion was further homogenized with 2% w/v PVA solution as a co-surfactant to form a double emulsion (w/o/w). The prepared AO-SLNs were stored at 4°C until further characterization²³.

 

Nanoparticle characterization:

Transmission electron microscopy (TEM):

The surface morphology of AO-SLNs was observed using a JEOL 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan)²⁴.

 

Particle size analysis:

The particle size of the AO-SLNs was determined using a Malvern particle size analyser (Zetasizer 4000S, Japan)²⁴.

 

Polydispersity index (PDI):

The PDI was determined to evaluate the monodisperse or polydisperse nature of the prepared nanoparticles. Higher values of the PDI indicate greater non-uniformity ²⁵. All analyses were carried out in triplicate.

 

Zeta potential (ZP) measurements:

The ZP of the AO-SLNs was measured using a Malvern instrument 3000HSA, UK²⁶.

 

Encapsulation efficiency (EE):

To calculate the amount of AO entrapped (EE) inside the prepared nanoparticles, gravimetric technique was used ²⁷. Approximately 100 mg of the different prepared SLN formulations (F1-F6) was dissolved in 25ml of dichloromethane (DCM) and then centrifuged at 12,000 rpm. The obtained supernatants for the formulations were introduced into separate Clevenger apparatuses with 50ml flat-bottomed flasks. Boiling clips were added, and the solutions were distilled for 1 h. The volume of AO in each formulation was directly read from the oil collection arm and multiplied by the density of the oil system to calculate the amount of AO entrapped in each formulation in (g). Samples were run in triplicate, and the results are tabulated in (Table 4).

 

Table 1. Formulations of AO-SLNs

Ingredients	F1	F2	F3	F4	F5	F6
Aegle oil (ml)	1	1	1	1	1	1
Lecithin (mg)	25	50	100	25	50	100
Compritol 888 (mg)	10	10	10	-	-	-
Cholesterol (mg)	-	-	-	10	10	10
Tween 80	2%	3%	4%	2%	3%	4%
PVA	2%	2%	2%	2%	2%	2%
Distilled water	QS	QS	QS	QS	QS	QS

 

Drug diffusion study:

The in vitro release of AO from different prepared SLN formulations (F1-F6) was assessed using a modified Franz diffusion cell²⁸.

 

Preparation of a topical AO hydrogel:

The optimized AO-SLN formulation (F6) was prepared as a topical hydrogel using 1% Carbopol 934 as a gelling agent.

 

Evaluation of topical AO-SLN hydrogel:

The physical appearance, pH and rheological properties of the AO-SLN hydrogel were evaluated.

 

Physical examination and pH determination:

The prepared AO gel was inspected visually to determine its colour, homogeneity, consistency, spreadability, phase separation²⁹ and pH determination using a previously calibrated pH meter^31.

 

Antimicrobial screening:

The antibacterial and antifungal activities of the leaves, EO, SLN, SLN gel were tested using the disc diffusion method against gram-positive bacteria (Bacillus subtilis (RCMB 010067) and Staphylococcus aureus (RCMB 010010)), gram-negative bacteria (Escherichia coli (RCMB 010052)) and Pseudomonas aeruginosa (RCMB 010043)), and fungi (Candida albicans (RCMB 05036), and Aspergillus fumigatus (RCMB 02568)). Filter paper discs (6 mm) were impregnated with the test substances and compared with positive control (ampicillin, gentamicin and amphotericin B)³⁰. The plates were left for 1 h at room temperature as a period of pre-incubation diffusion and then incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. The diameters of the inhibition zones were measured in millimetres.

 

Determination of the minimum inhibitory concentration (MIC):

The MIC was determined by the broth microdilution method using 96-well microplates³¹. Plates were incubated at 37°C for 24 h to determine antibacterial activity and at 25°C for 48 h to determine antifungal activity.

 

RESULTS AND DISCUSSION:

Characterization of A. marmelos essential oil:

A. marmelos leaf essential oil showed a predominance of monoterpenes, representing 97.12% of the identified content, whereas Δ-carene represented 48.14%, followed by α-phellandrene (34.14%) and α-pinene (6.81%) (Table 2). Similar results were previously reported in the Egyptian variety but with different percentages, which is probably due to seasonal variation¹². Additionally, α-phellandrene was previously reported to alter the morphology of P. cyclopium hyphae by causing loss of cytoplasmic material and distortion of the mycelia³². α-and β-pinene have also demonstrated antimicrobial activity beside promising effect against biofilm formation³³

 

Table 2. Chemical compositions of A. marmelos leaf essential oil

Name	KI	Area %
α-Thujene	945.736	0.03
α-Pinene	954.511	6.81
α-Fenchene	974.198	0.05
Sabinene	1008.45	0.31
β-Pinene	1012.55	0.05
Myrecene	1032.81	2.69
α-Phellandrene	1052.86	34.14
β-Phellandrene	1059.23	0.15
p-Cymene	1079.76	3.03
Δ-Carene	1087.69	48.14
Z-β-Ocimene	1098.11	0.25
E-β-Ocimene	1112.32	1.44
ɣ-Terpinene	1127.28	0.03
Terpinolene	1168.84	0.16
Linalyl acetate	1185.1	0.23
p-Mentha-2,8-dien-1-ol	1255.34	0.04
α-Limonene diepoxide	1394.08	0.44
Isothujol	1478.75	0.11
Carveol	1573.76	0.06
Caryophyllene	1606.85	0.07
Germacrene B	1620.93	0.11
β-Curcumene	1673.44	0.07
ɣ-Elemenene	1767.51	1.06
% of identified compounds		99.31
% of monoterpenes		97.12
% of oxygenated monoterpenes		0.88
% sesquiterpenes		1.31

 

Estimation of the total phenolic content:

The content of total phenols was measured by FCR in terms of GAE (standard curve equation: y = 0.0011x + 0.0009, r2 = 0.9867). The phenolic content was estimated to be 968mg/g GAE.

 

UPLC-ESI-MS-MS analyses:

UPLC-ESI-MS-MS analyses of A. marmelos methanolic leaf extract led to the tentative characterization of thirteen metabolites belonging to different classes based on comparisons of their MS/MS spectra with the available literature (Table 3). The analyses were performed in both positive and negative mode, which enabled the determination of the exact m/z values of [M+H]⁺ and [M-H]^- and product ion fragments for all assigned metabolites³⁴.

 

Table 3. Peak assignment of metabolites in A. marmelos leaf methanolic extract using UPLC-ESI-MS-MS

No.	Molecular weight	Molecular ion (m/z)	MS/MS (m/z)	Tentative identification	Ref.
Alkaloids
1	297	298 [M+H]+	280, 190, 171, 117, 105, 84, 45	Aegeline	35
2	324	325 [M+H]+	206, 181, 175, 111, 87, 45	N-2-Ethoxp-2-(4-methxyphenylethylcinnamide	8,36
3	459	460 [M+H]+	325, 296, 173, 163, 134, 117, 88	Aegelinoside A	37
Coumarins
4	259	260 [M+H]+	258, 228, 212, 186, 168, 166, 141, 69	Marmelonine	8
5	277	278 [M+H]+	256, 246, 232, 183, 174, 149	(20S,30R)-8-Hydroxysmyrindiol	8
6	270	271 [M+H]+	241, 227, 142, 81, 43	Marmelosin	38
7	216	217 [M+H]+	202, 185, 173, 145, 105, 87, 81, 73	Bergapten	38
8	287	288 [M+H]+	229, 224, 182, 177, 114, 89, 69	6-(4-Acetoxy-3-methyl-2-butenyl)-7-hydroxycoumarin	38
9	298	299 [M+H]+	266, 245, 219, 203, 175, 169, 145, 126, 105	Auraptene	39,40
10	246	247 [M+H]+	230, 201, 185, 177, 131, 129, 113, 105, 83, 69, 45	Marmesin	39,40
11	202	203 [M+H]+	185, 157, 139, 111, 85	Xanthotoxol or 8-OH-psoralen	39,40
Flavonoids
12	448	449 [M+H]+	285, 183	Kaempferol dihydroglucoside
13	289	290 [M+H]+	275, 205, 162, 154, 136, 85	(+)-4-(20-Hydroxy-30-methylbut-30-enyloxy)-8H-[1,3] dioxolo[4,5-h]chromen-8-one

 

Nanoparticles characterization:

Transmission electron microscope (TEM):

Regardless of the type of lipid used in the experiment, TEM revealed that AO-SLNs were nanometre-sized with a good size distribution. The round shapes observed ensure the malleability of the formed vesicles. The TEM observations were in agreement with the Malvern size measurements (Fig 1).

 

Figure 1. Transmission electron micrograph of AO-SLNs

 

Particle size analysis, PDI and ZP measurements:

The particle size of all AO formulations (F1- F6) is presented as the z-average diameter between 111 - 220 nm. Likewise by increasing the concentration of surfactant, the mean particle size of the SLNs was reduced enough to coat all the lipid droplets, while the organic solvent used in these formulations rapidly partitioned into the continuous aqueous medium, and the lipid precipitated around the drug. PDI of the AO-SLNs ranged between 0.43-0.79, which indicates a uniform particle size and stability of the prepared nanoparticles. The PDI was reduced to a narrow range by increasing the surfactant concentration. Furthermore, All AO-SLN formulations showed negative charged values, which indicates long-term physical stability and particle adhesion properties. All results of the previously investigated parameters are tabulated in (Table 4).

 

Entrapment efficiency (EE):

Referring to Table 4, the EEs for all the AO-SLN formulations were promising, ranging from 65.10 to 91.90%. The EE was expected to increase with an increasing lipid concentration. Formulation F6 showed the highest efficiency of 91.90%, as it provided more space for AO encapsulation.

 

Drug diffusion study:

The in vitro release profiles of AO-SLN formulations containing different lipid (lecithin) concentrations were evaluated using a modified Franz diffusion cell (Fig 2). The prepared nanoparticles showed an initial burst release (40 min) of 5.56% - 40.05%. F6, with a small particle size, showed less drug release due to its highly polymeric matrix. At the end of 12 h, a limited percentage (approximately 85.54%) of drug release was observed.

 

 

Table 4. Evaluation of different AO-SLN formulations

Formulations	Particle size (nm) ± SD	PDI (%) ± SD	Zeta potential (mv)	% Entrapment efficiency ± SD
F1	200 ± 0.26	0.686 ± 0.56	-30	65.10 ± 0.75
F2	188 ± 0.32	0.511 ± 0.47	-35	75.05 ± 0.98
F3	145 ± 0.42	0.433 ± 0.76	-33	88.25 ± 0.45
F4	220 ± 0.12	0.791± 0.12	-31	66.05 ± 0.26
F5	165 ± 0.25	0.476 ± 0.34	-33	79.50 ± 0.64
F6	111 ± 0.42	0.431 ± 0.56	-40	91.90 ± 0.87

(Mean ± SD, n=3)

 

Figure 2. In vitro diffusion of different AO-SLNs formulations

 

Evaluation of the topical AO-SLN hydrogel:

To investigate the antimicrobial activity of AO, the optimized AO-SLN formulation (F6) was prepared as a topical hydrogel using 1% Carbopol 934 as a gelling agent. Visual inspection of the prepared hydrogel indicated suitable homogeneity and consistency, with no lumps or phase separation with a pH of 6.2 ± 0.68, providing an acceptable range to avoid dermal irritation following application.

 

Antimicrobial screening and MIC evaluation for essential oil and hydrogel formulation:

AO showed the best antimicrobial activity with inhibition zone varying from 11 to 20mm compared to 9 and 11mm for the leaf extract (Table 5). Those results were in agreement with previous reports¹². The MIC values were estimated for the leaf oil as 11, 14 and 18 µg/ml against S.aureus, E.coli and C.albicans respectively (Table 6). Those results were compared to the prepared AO-SLNs and the gel formulation (Table 6). A 50% decrease in the MICs was observed for all the tested microorganisms, highlighting the ability of nanoparticles to enhance essential oil antimicrobial activity, providing higher stability and solubility, and enabling drug targeting^2,5.

Table 5. Antimicrobial activity of the leaves and leaf essential oil expressed as the inhibition zone diameter [mm]^a

Tested organisms	Leaf extract	Leaf essential oil	Standards
Gram-positive			Ampicillin
Bacillus subtilis (RCMB 010067)	10±0.2	15±0.15	32±0.23
Staphylococcus aureus (RCMB010010)	11±0.12	20±0.18	24±0.21
Gram-negative			Gentamicin
Escherichia coli (RCMB 010052)	9±0.17	17±0.2	20±0.15
Pseudomonas aeruginosa (RCMB 010043)	9±0.2	11±0.13	17±0.15
Fungi			Amphotericin B
Candida albicans (RCMB 05036)	-	18±0.15	25±0.14
Aspergillus fumigatus (RCMB 02568)	-	12±0.2	24±0.14

Mean zone of inhibition in mm ± standard deviation beyond the well diameter (6 mm).

 

Table 6. Antimicrobial activity as the MIC [µg/ml] of the essential oil from leaves, SLN and SLN hydrogel

Tested organisms	Leaf oil	SLN	SLN hydrogel	Standard
Gram-positive				Ampicillin
Staphylococcus aureus	11	8	5	0.49
Gram-negative				Gentamicin
Escherichia coli (RCMB 010052)	14	9	7	0.98
Fungi				Amphotericin B
Candida albicans (RCMB 05036)	18	10	9	0.49

 

CONCLUSION:

In the present study, in-vitro assessment of AO formulated as SLN hydrogel showed enhanced activity against C.albicans and S.aureus. Therefore providing an evidence for its use in topical and transdermal delivery systems.

 

CONFLICT OF INTEREST:

The authors declare there are no conflicts of interest.

 

REFERENCES:

1.      Fallah Huseini H, Fakhrzadeh H, Larijani B, Shikh Samani A. Review of anti-diabetic medicinal plant used in traditional medicine. J Med Plants. 2006;1(17): 1-8.

2.      Pedro a S, Santo IE, Silva C V, Detoni C, Albuquerque E. Microbial pathogens and strategies for combating them: science, technology and education. In: Méndez-Vilas A, ed. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. Formatex; 2013: 1364-1374.

3.      Cavalli R, Caputo O, Gasco MR. Preparation and characterization of solid lipid nanospheres containing paclitaxel. Eur J Pharm Sci. 2000;10: 305-309.

4.      Allahverdiyev AM, Kon KV, Abamor ES, Bagirova M, Rafailovich M. Coping with antibiotic resistance: Combining nanoparticles with antibiotics and other antimicrobial agents. Expert Rev Anti Infect Ther. 2011; 9: 1035-1052.

5.      Zhaveh S, Mohsenifar A, Beiki M, et al. Encapsulation of Cuminum cyminum essential oils in chitosan-caffeic acid nanogel with enhanced antimicrobial activity against Aspergillus flavus. Ind Crops Prod. 2015;69: 251-256.

6.      Najafi-taher R, Ghaemi B, Kharrazi S, Rasoulikoohi S, Amani A. Promising antibacterial effects of silver nanoparticle-loaded tea tree oil nanoemulsion: a synergistic combination against resistance threat. AAPS Pharm SciTech. 2018;19(3): 1133-1140. doi:10.1208/s12249-018-0950-2

7.      Manandhar B, Paudel KR, Sharma B, Karki R. Phytochemical profile and pharmacological activity of Aegle marmelos Linn. J Integr Med. 2018; 16 (3): 153-163.

8.      Chakthong S, Weaaryee P, Puangphet P, et al. Alkaloid and coumarins from the green fruits of Aegle marmelos. Phytochemistry. 2012; 75: 108-113.

9.      Mishra BB, Kishore N, Tiwari VK, Singh DD, Tripathi V. A novel antifungal anthraquinone from seeds of Aegle marmelos Correa (family Rutaceae). Fitoterapia. 2010;81(2): 104-107. doi:10.1016/ j.fitote.2009.08.009

10.   Mishra BB, Singh DD, Kishore N, Tiwari VK, Tripathi V. Antifungal constituents isolated from the seeds of Aegle marmelos. Phytochemistry. 2010;71(2-3): 230-234. doi:10.1016/ j.phytochem.2009.10.013

11.   Sain S, Naoghare P, Devi S, et al. Beta Caryophyllene and Caryophyllene Oxide, Isolated from Aegle Marmelos, as the Potent Anti-inflammatory Agents against Lymphoma and Neuroblastoma Cells. Antiinflamm Antiallergy Agents Med Chem. 2014;13(1):45-55. doi:10.2174/18715230113129990016

12.   Ibrahim NA, El-Sakhawy FM, Mohammed MMD, Farid MA, Abdel-Wahed NAM, Deabes DAH. Chemical composition, antimicrobial and antifungal activities of essential oils of the leaves of Aegle marmelos (L.) Correa growing in Egypt. J Appl Pharm Sci. 2015; 5(2): 1-5.

13.   Jamal MAHM, Rahman MS, Hossain MB, et al. Antibacterial properties and chemical composition of essential oil from Aegle marmelos (L.) Corr. leaves growing in Bangladesh. J Essent Oil-Bearing Plants. 2017;20(1): 155-174. doi:10.1080/0972060X.2017.1308276

14.   Gandhi GR, Ignacimuthu S, Paulraj MG. Hypoglycemic and β-cells regenerative effects of Aegle marmelos (L.) Corr. bark extract in streptozotocin-induced diabetic rats. Food Chem Toxicol. 2012;50(5): 1667-1674.

15.   Karmase A, Birari R, Bhutani KK. Evaluation of anti-obesity effect of Aegle marmelos leaves. Phytomedicine. 2013;20(10): 805-812.

16.   Pynam H, Dharmesh SM. Antioxidant and anti-inflammatory properties of marmelosin from Bael (Aegle marmelos L.); Inhibition of TNF-α mediated inflammatory/tumor markers. Biomed Pharmacother. 2018; 106:98-108.

17.   Rathee D, Kamboj A, Sachdev RK, Sidhu S. Hepatoprotective effect of Aegle marmelos augmented with piperine co-administration in paracetamol model. Brazilian J Pharmacogn. 2018; 28: 65-72.

18.   Modi H, Patel V, Patel K. Hepatoprotective activity of Aegle marrmelos against ethanol induced hepatotoxicity in rats. Asian J Pharm Clin Res. 2012;5: 164-167.

19.   Mohammed MMD, Ibrahim NA, El-Sakhawy FS, Mohamed KM, Deabes DA-H. Two new cytotoxic furoquinoline alkaloids isolated from Aegle marmelos (Linn.) Correa. Nat Prod Res. 2016;30(22): 2559-2566.

20.   Shahat EA, Bakr RO, Eldahshan OA, Ayoub NA. Chemical Composition and Biological Activities of the Essential Oil from Leaves and Flowers of Pulicaria incisa sub. candolleana (Family Asteraceae). Chem Biodivers. 2017; 14(4): e1600156

21.   Adams RP. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Pub. Corp; 2007.

22.   Sellappan S, Akoh CC, Krewer G. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J Agric Food Chem. 2002;50(8): 2432-2438.

23.   Trotta M, Cavalli R, Carlotti ME, Battaglia L, Debernardi F. Solid lipid micro-particles carrying insulin formed by solvent-in-water emulsion-diffusion technique. Int J Pharm. 2005;288: 281-288.

24.   Shekhawat PB. Preparation and evaluation of Clotrimazole nanostructured lipid carrier for topical delivery. Int J Pharma Bio Sci. 2012;4(1): 407-416.

25.   Yener G, Incegül T, Yener N. Importance of using solid lipid microspheres as carriers for UV filters on the example octyl methoxy cinnamate. Int J Pharm. 2003;258: 203-207.

26.   Praveen S, Gowda D V., Srivastava A, Osmani RAM. Formulation and evaluation of nanostructured lipid carrier (NLC) for glimepiride. Der Pharm Lett. 2016;8(7): 251-256.

27.   Vishwakarma GS, Gautam N, Babu JN, Mittal S, Jaitak V. Polymeric Encapsulates of Essential Oils and Their Constituents: A Review of Preparation Techniques, Characterization, and Sustainable Release Mechanisms. Polym Rev. 2016;56(4): 668-701.

28.   Wissing SA, Muller RH. Solid lipid nanoparticles as carrier for sunscreens: in vitro release and in vivo skin penetration. J Control Release. 2002;81: 225-233.

29.   Mohamed MI. Optimization of chlorphenesin emulgel formulation. AAPS J. 2004;6(3): 1-7.

30.   Mickymaray S, Al Aboody MS, Rath PK, Annamalai P, Nooruddin T. Screening and antibacterial efficacy of selected Indian medicinal plants. Asian Pac J Trop Biomed. 2016; 6(3): 185-191.

31.   Saini KR, Choudhary SA, Joshi YC, Joshi P. Solvent free synthesis of chalcones and their antibacterial activities. E-Journal Chem. 2005;2(4):224-227. doi:10.1155/2005/294094

32.   Zhang J hong, Sun H long, Chen S yang, Zeng LI, Wang T tao. Anti-fungal activity, mechanism studies on α-Phellandrene and Nonanal against Penicillium cyclopium. Bot Stud. 2017; 58:13.

33.   Da Silva ACR, Lopes PM, De Azevedo MMB, Costa DCM, Alviano CS, Alviano DS. Biological activities of α-pinene and β-pinene enantiomers. Molecules. 2012;17(6): 6305–6316.

34.   Ibrahim NA, Mohammed MMD, Aly HF, Ali SA, Al-Hady D-A. Efficiency of the leaves and fruits of Aegle marmelos methanol extract (L.) Correa and their relative hepatotoxicity induced by CCL4 and identification of their active constituents by using LC/MS/MS. Toxicol Reports. 2018; 5: 1161–1168.

35.   Nugroho AE, Riyanto S, Sukari MA, Maeyama K. Effects of aegeline, a main alkaloid of Aegle marmelos correa leaves, on the histamine release from mast cells. Pak J Pharm Sci. 2011; 24(3): 359-367.

36.   Manandhar MD, Shoeb A, Kapil RS, Popli SP. New alkaloids from Aegle marmelos. Phytochemistry. 1978;17(10): 1814-1815.

37.   Phuwapraisirisan P, Puksasook T, Jong-aramruang J, Kokpol U. Phenylethyl cinnamides: A new series of α-glucosidase inhibitors from the leaves of Aegle marmelos. Bioorganic Med Chem Lett. 2008;18(18): 4956-4958.

38.   Shinde PB, Laddha KS. Simultaneous quantification of furanocoumarins from Aegle marmelos fruit pulp extract. J Chromatogr Sci. 2015; 53(4): 576-579.

39.   P. Santhana Krishnan V, Karthikeyan G, Janaki P, Poonkodi B, Sathya R. Isolation of Heraclenin from Aegle marmelos correa and screening for its antimicrobial activity through in vitro & in silico studies. Nat Prod J. 2016;6(2): 134-141.

40.   Ali MS, Pervez MK. Marmenol: A 7-geranyloxycoumarin from the leaves of Aegle marmelos corr. Nat Prod Res. 2004; 18(2): 141-146.

 

 

 

Received on 09.09.2020           Modified on 11.10.2020

Accepted on 29.10.2020         © RJPT All right reserved