Phytochemical analyses and pharmacological screening of Neem oil
S. Cesa
a,⁎
, F. Sisto
b, G. Zengin
c, D. Scaccabarozzi
d, A.K. Kokolakis
e, M.M. Scaltrito
b, R. Grande
f, M. Locatelli
f,
F. Cacciagrano
f, L. Angiolella
g, C. Campestre
f, A. Granese
a, P. Chimenti
a, N. Basilico
ba
Dipartimento di Chimica e Tecnologie del Farmaco,“Sapienza” Università di Roma, Rome, Italy
b
Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, University of Milan, Milan, Italy
c
Department of Biology, Science Faculty, Selcuk University, Konya, Turkey
d
Dipartimento di Scienze Farmacologiche e Biomolecolari, University of Milan, Milan, Italy
e
Department of Chemistry, University of Crete, Heraklion, Greece
fDepartment of Pharmacy and Center of Aging Sciences and Translational Medicine (CeSI-MeT), University“G. d'Annunzio” of Chieti-Pescara, Chieti, Italy g
Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy
a b s t r a c t
a r t i c l e i n f o
Article history: Received 8 May 2018
Received in revised form 12 September 2018 Accepted 25 October 2018
Available online 15 November 2018 Edited by KRR Rengasamy
An Italian certified Neem seed oil was characterized through the color analysis, the HPLC phenolic fingerprint and the preliminary evaluation of the cytotoxicity profile against the human macrophage (THP-1) cell line. Moreover, a wide screening of its enzyme inhibitory profile, antimicrobial activity towards Helicobacter pylori, several Candida spp. and Malassezia furfur strains and antiprotozoal activity against Plasmodium falciparum, Leishmania infantum and Leishmania tropica were performed. Neem seed oil demonstrated low toxicity and a great inhibitory efficacy against tyrosinase and lipase enzymes. Antimalarial and anti-leishmanial activities were also demon-strated, but weak or no activity was evidenced against Helicobacter pylori, Candida and Malassezia strains. Overall, thesefindings encourage the potential use of this natural product in some disease treatments and justify its use in traditional medicine.
© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords: Color analysis HPLC-PDA H. pylori Neem oil Malaria Leishmania Enzyme inhibition 1. Introduction
Azadirachta indica A. Juss (Meliaceae), best known as Neem, repre-sents the subject of extensive investigations for its numerous biological activities. The oil obtained from Neem seeds, in which triterpenoids, limonoids and alkaloids were recognized, preserves many of the phar-macological properties expressed by the whole plant or by its parts. Extracts from Neem leaves, fruits and seeds, containing approximately 0.13% oil, have been used from ancient times in pharmaceutical preparations.
It was demonstrated that Neem seeds contain a large plethora of constituents, which influence the biological activity. According to
Kumar and Parmar (1996), neem kernel is characterized by a high amount of oil (up to 52% w/w). In terms of fatty acid composition, this commercial Neem oil is mainly composed of oleic (58%), palmitic (14%) and stearic (15%) acids, whereas myristic, arachidic, linoleic
and behenic acids are detectable only in small amounts. In the obtained oil, triterpenoids such as salannin, nimbin and azadirachtin, known for their insecticide properties, are also present along with sterols. All these molecules are recognized as biologically active ingredients (Pandey et al., 2014). Nimbin, accounting for much of the bi-ological activities of Neem oil, shows anti-inflammatory, antipyretic, fungicidal, antihistamine and antiseptic properties (Gupta et al., 2017). Azadirachtin, a powerful acetylcholinesterase inhibitor (Nathan et al. 2008), and salannin are undergoing evaluation for their NO-production inhibitory activity (Akihisa et al., 2017). More than 140 com-pounds have been isolated from all parts of the Neem tree (flowers, leaves, seeds, fruits, roots, bark), to which interferon inducing activity (bark), immunomodulatory, antipyretic and anti-inflammatory, antiul-cer, antimalarial, antifungal, antibacterial, antiviral against skin ailments activity (leaves), as well as antioxidant and antimutagenic properties were recognized (SaiRam et al., 2000; Subapriya and Nagini, 2005).
Well-known are the secondary metabolites, derived from higher plants, which act as efficacious defense agents against microorganisms and are under study for their antimicrobial properties. The antimicrobial resistance, and in particular the multidrug resistance, represents a topic of primary interest for the worldwide scientific community and new
⁎ Corresponding author at: Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le Aldo Moro, 5 - 00185 Rome, Italy.
E-mail address:stefania.cesa@uniroma1.it(S. Cesa).
https://doi.org/10.1016/j.sajb.2018.10.019
0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
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therapeutic strategies, in answer to this growing and worrying phe-nomenon, are extensively researched. In fact, in the last decades, it has been recorded an increasing number of multidrug resistance pathogens, because of an indiscriminate use of antibiotic therapies.
The antimicrobial activity of Neem oil was evaluated as a synergistic interaction with other essential oils, which combine terpenoid com-pounds, demonstrating that the presence of Neem oil confers a broad spectrum of antibacterial activity (Kurekci et al., 2013). Additive and synergistic effects were also shown byGovindachari et al. (1998)
which reported that the isolated triterpenoids had very low or no activ-ity, whereas in combination they exerted excellent activity against dif-ferent phytopathogenic fungi (Ali et al., 2017). A more recent study on Staphylococcus and Salmonella spp. confirmed the strong potential of this natural product for the explicated antibacterial activity (Zhang et al., 2010).
In the tropics, A. indica has been used commonly as a traditional an-timalarial preparation (Phillipson and Wright, 1991; Leaman et al., 1995) and several in vitro and in vivo studies confirmed that the leaf and seed extracts possess inhibitory activity against both sexual and asexual stages of Plasmodium falciparum (Udeinya et al., 2008; Lucantoni et al., 2010). In particular, nimbolide, gedunin and epoxyazadiradione seem to be the main metabolites responsible for an-timalarial activity (Biswas et al., 2002; Chianese et al., 2010). Con-versely, the activity of Neem oil against Leishmania spp. is less documented, with the anti-leishmanial effects of leaf extracts against L. donovani reported byDayakar et al. (2015).
Many plants provide different interesting classes of compounds, such as alkaloids, tannins, quinones, polypeptides,flavonoids, couma-rins, terpenoids, characterized by a high potential to limit bacterial, fun-gal, protozoal and viral diseases. Among these plants, Azadirachta indica, endemic of India, which grows in several Asian and African countries, but in America and Australia as well, was investigated for its anti-inflammatory, anticancer, antidiabetic, antihypertensive and neuropro-tective activity (Gupta et al., 2017).
For the promising activity in oral hygiene and against skin parasites, barks, leaves and seed oil from this plant were traditionally used in Ayurvedic medicine, the herbal medicine that is the only choice for India's rural areas. Only few studies are available in literature regarding the total phenolic and totalflavonoid content of Neem oil beyond the li-monoid content (Naseer et al., 2014; Singh et al., 2005).
On the other hand, different experiments carried on with the objec-tive to evaluate the enzyme inhibitory activity (Mukherjee and Sengupta, 2013; Manosroi et al., 2014), demonstrated the potential of some limonoid andflavonoid molecules from seeds to be used as antidi-abetic drugs or as melanogenesis inhibitors. Over the last decade, the prevalence of some diseases including Alzheimer's disease, diabetes mellitus and obesity has dramatically increased and millions of peoples are affected by them. Considering this, effective therapeutic strategies are urgently required to control these diseases. Among these strategies, the key enzyme inhibitory theory could be considered one of the most useful approaches to manage health problems on a global scale. The main target with key enzyme inhibition is to alleviate the symptoms of these diseases. In this concept, several synthetics were designed as in-hibitors but they gave rise to problems such as hepatotoxicity and gas-trointestinal disturbances. This phenomenon stimulates the discovery of novel, effective and safer inhibitors from natural sources (Zengin et al., 2018).
On the basis of these premises and pursuing our research studies on this natural product (Carradori et al., 2015; Rinaldi et al., 2017), an Ital-ian certified Neem seed oil was further characterized in the phytochem-ical composition (color analysis and HPLC phenolicfingerprint) and submitted to a screening of its biological properties in relation to the en-zyme inhibitory activity towards cholinesterases (AChE and BChE), α-amylase,α-glucosidase, tyrosinase and pancreatic lipase. The antibacte-rial activity, against 8 highly resistant Helicobacter pylori strains, the an-tifungal activity, against 26 Candida spp. and 20 Malassezia furfur strains
and the antiprotozoal activity against P. falciparum, L. infantum and L. tropica were evaluated with the objective to better understand the wide-ranging activity of Neem seed oil. Moreover, a preliminary evalu-ation of the cytotoxicity profile of this oil was assessed against the human macrophage (THP-1) cell line, because of the reported evidence that oral ingestion could be associated with encephalopathy in adult men, liver damage in children and certain damage to pregnant women (Boeke et al., 2004).
2. Material and methods 2.1. Chemicals
Neem oil was purchased by Neem Italia (Italy) and was authenti-cated and characterized by the ECOCERT certificate (Biocert Italia IT013BC041– ICEA 264BC001). All chemicals used for the HPLC-PDA analyses (gallic acid, catechin, chlorogenic acid, p-hydroxybenzoic acid, vanillic acid, epicatechin, syringic acid, hydroxybenzoic acid, 3-hydroxy-4-methoxybenzaldehyde, p-coumaric acid, rutin, sinapinic acid, t-ferulic acid, naringin, 2,3-dimethoxybenzoic acid, benzoic acid, o-coumaric acid, quercetin, harpagoside, t-cinnamic acid, naringenin, and carvacrol (all purity 98%)) and for the biological assays as well as methanol and acetonitrile (HPLC-grade) were obtained from Sigma-Aldrich (Milan, Italy), while HPLC-grade acetic acid was bought from Carlo Erba Reagents (Milan, Italy). Double distilled water (Milli-Q sys-tem, Millipore, Bedford, USA) was used.
2.2. Color analysis
Colorimetric CIELAB parameters, (L*, a*, b*, C*aband hab), as defined by the“Commission Internationale de l'Eclairage”, were determined on the seed oil of Azadirachta indica using a colorimeter X-Rite SP-62 (X-Rite Europe GmbH, Regensdorf, Switzerland), equipped with a D65 illuminant and an observer angle of 10°. The color description is based on three parameters: L* defining the lightness and varying between 0 (absolute black) and 100 (absolute white), a* measuring the greenness (−a*) or the redness (+a*) and b* evaluating the blueness (−b*) and the yellowness (+b*). C*ab(chroma, saturation) expresses a measure of color intensity, whereas hab(hue, color angle) is the attribute of ap-pearance by which a color is identified according to its resemblance to red, yellow, green or blue, or to a combination of two of these character-istics in sequence. Cylindrical coordinates C*aband habwere calculated from the parameters a* and b* using the eqs. C*ab= (a*2+ b*2)½and hab= tan−1(b*/a*) (Clydesdale and Ahmed, 1978). The results are expressed as the mean value ± standard deviation (SD) of at least four different experiments.
2.3. HPLC analysis
Neem oil was analyzed for the multicomponent pattern quantitative determination of important and abundant phenols andflavonoids, after being diluted with n-hexane in ratio 1:5 and 1:20 (v:v), by means of a reversed phase HPLC-PDA in gradient elution mode. Analyses were car-ried out by using a liquid chromatography system (Waters SpA, Milan, Italy) equipped with a photodiode array detector, a C18 reversed-phase column (Prodigy ODS(3), 4.6 × 150 mm, 5μm; Phenomenex, Torrance, CA), an online degasser (Biotech 4-CH degasi compact, LabService, Anzola Emilia, Italy), a column oven set at 30 °C (± 1 °C). The gradient elution was achieved using a solution of water–acetonitrile (93:7 v:v ratio, with 3% of acetic acid) as initial settings, and the com-plete separation was achieved in 60 min according to a validated method (Locatelli et al., 2017) and herein applied after evaluation of the analytical performances to assess the absence of matrix interfer-ences (Supplementary data). Data were reported as mean values ± SD of three independent determinations and were expressed asμg/mL of Neem oil.
2.4. Enzyme inhibitory activity
The enzyme inhibitory activities of the oil were obtained as equiva-lents of standard drugs per gram of the oil sample (galantamine for AChE and BChE, kojic acid for tyrosinase, orlistat for lipase, and acarbose forα-amylase and α-glucosidase inhibition assays).
2.4.1. Cholinesterase inhibition
Test solution (50μL) was mixed with DTNB (5,5′-dithiobis(2-nitrobenzoic acid) (125μL)) and enzyme (AChE or BChE) solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at room temperature. The reaction was then started with the addition of the corresponding substrates (acetylthiocholine iodide or butyrylthiocholine chloride) (25μL). Similarly, a blank was prepared without the enzyme (AChE or BChE) solution. The absorbance of sample and blank were registered at 405 nm after 10 min of incubation at 25 °C. Results were given as milligrams of galantamine equivalents per gram of oil (mg GALAEs/g oil) (Zengin et al., 2016a).
2.4.2.α-Amylase inhibition
The test solution (25μL) was mixed with α-amylase solution (50 μL) in a phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After this incubation, the reaction was started with the addition of starch solution (50μL, 0.05%). Similarly, a blank (without the enzyme solution) was prepared. The re-action was stopped with the addition of HCl (25μL, 1 M) and then the iodine-potassium iodide solution was added (100μL). The absorbance of sample and blank were evaluated at 630 nm. Results were given as millimoles of acarbose equivalents per gram of oil (mg ACAEs/g oil) (Zengin et al., 2016b).
2.4.3.α-Glucosidase inhibition
The test solution (50μL) was mixed with glutathione (50 μL), α-glucosidase solution (50μL) in phosphate buffer (pH 6.8) and PNPG (p-nitrophenyl-β-D-glucuronide, 50 μL) in a 96-well microplate and in-cubated for 15 min at 37 °C. Similarly, a blank (without the enzyme so-lution) was prepared. The reaction was stopped with sodium carbonate (50μL, 0.2 M). The absorbance of sample and blank were noted at 400 nm. The results were expressed as millimoles of acarbose equiva-lents per gram of oil (mmol ACAEs/g oil) (Zengin et al., 2016b). 2.4.4. Tyrosinase inhibition
The test solution (25μL) was mixed with tyrosinase solution (40 μL) and phosphate buffer (100μL, pH 6.8) in a 96-well microplate and incu-bated for 15 min at 25 °C. The reaction was then started with the addi-tion of the L-DOPA soluaddi-tion (40 μL). Similarly, a blank was done (without the enzyme solution). The absorbance of sample and blank were recorded at 492 nm after 10 min of incubation at 25 °C. The results were expressed as milligrams of kojic acid equivalents per gram of oil (mg KAE/g oil) (Zengin et al., 2016b).
2.4.5. Lipase inhibition
Porcine pancreatic lipase (type-II) activity was performed using p-nitrophenyl butyrate (p-NPB) as substrate (Roh and Jung, 2012). The enzyme solution (1 mg/mL) was prepared in 50 mM Tris–HCl (pH 8.0). An aliquot of this solution (25μL) was mixed with the lipase solution (50μL) in a 96-well microplate and incubated for 20 min at 25 °C. The reaction was started with the addition of p-NPB solution (5 mM, 50μL). Similarly, a blank sample (without the enzyme) was pre-pared and analyzed in accordance with this procedure. Milligrams of orlistat equivalents per gram of oil (mg OEs/g oil) were the measure unit. 2.5. Helicobacter pylori strains culture
Seven clinical isolates of H. pylori, having a different susceptibility pattern against commercially available antibiotics, were considered
including four resistant strains to metronidazole (MNZ) (MICN 8 μg/ mL), four resistant strains to clarithromycin (CLR) (MICN 0.5 μg/mL), two resistant strains to both MNZ and CLR, and two CLR, MNZ and amoxicillin (AMX) susceptible strains (MIC≤0.125 μg/mL). A reference strain of H. pylori (NCTC 11637) was also used as a control for this eval-uation. The strains were maintained at−80 °C in Wilkins Chalgren Broth (Difco, BD, San Jose, CA, USA) with 10% (v/v) horse serum (Seromed, Biochrom, Germany) and 20% (v/v) glycerol (Merck, Darm-stadt, Germany) until required for the experiments. Before being used, the strains were subcultured twice on Columbia agar base (Difco, BD, San Jose, CA, USA) supplemented with 10% horse serum and 0.25% bacto yeast extract (Difco, BD, San Jose, CA, USA). Plates were incubated for 72 h at 37 °C in microaerophilic conditions (10% CO2, 5% O2, 85% N2). The MICs were determined by the modified broth dilution method as previously described (Sisto et al., 2009). Neem oil was dissolved in DMSO, diluted with medium to achieve the required concentrations (final DMSO concentration b 0.02%, which is non-toxic to the bacte-rium). The dilutions of Neem oil ranged from 256 to 0.5μg/mL. Briefly, two-fold serial dilutions of the oil were prepared in a 96-well microtiter plates containing 100μL of MegaCell™ RPMI-1640 medium (Sigma-Al-drich, St. Louis, MO, USA) with 3% fetal calf serum (FCS). An inoculum equivalent to one McFarland standard was prepared in Wilkins Chalgren broth and diluted in MegaCell™ RPMI-1640 medium with 3% FCS. Each well was inoculated with H. pylori at afinal concentration of approximately 5 × 105CFU (Colony Forming Unit) × well. The plates were incubated at 37 °C under microaerophilic conditions and exam-ined after 72 h of incubation. For the MBC determination, aliquots (10 μL) of suspensions without visible growth were spotted on Columbia agar plates and incubated at 37 °C for 3–5 days under microaerophilic conditions. The MBC was determined as the lowest concentration of compound able to kill 99.9% of the microbial population.
2.6. Plasmodium falciparum cultures and drug susceptibility assay Plasmodium falciparum cultures were carried out according to Trager and Jensen with slight modifications (Trager and Jensen, 1976). The chloroquine (CQ)-susceptible strain D10 and the CQ-resistant strain W2 were maintained at 5% hematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone) medium with the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM Hepes, and 2 mM glutamine. All the cultures were maintained at 37 °C in a standard gas mixture consisting of 1% O2, 5% CO2, and 94% N2. Neem oil was dissolved in DMSO, diluted with medium to achieve the required concentrations (final DMSO concentration b 1%, which is non-toxic to the parasite), placed in 96-wellflat-bottomed microplates and serial dilutions were made. Asynchronous cultures with parasitaemia of 1–1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 h at 37 °C. Parasite growth was deter-mined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), in control and drug-treated cultures (De Monte et al., 2015). The antimalarial activity is expressed as 50% inhibitory concentration (IC50); each IC50value is the mean and SD of at least three separate experiments performed in duplicate. Chloroquine was used as the reference drug.
2.7. Evaluation of anti-leishmanial activity
The promastigote stages of L. infantum strain (MHOM/TN/80/IPT1) and of a clinical isolate of L. tropica (MHOM/IT/2012/ISS3130) were cul-tured in RPMI 1640 medium (EuroClone) supplemented with 10% heat-inactivated fetal calf serum (EuroClone), 20 mM Hepes, and 2 mM L-glutamine at 22 °C.
To estimate the 50% inhibitory concentration (IC50), the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method was used. Neem oil was dissolved in DMSO, diluted with medium to achieve the required concentrations, plated in 96 wells round-bottom
microplates and serial dilutions were made. Miltefosine was used as the reference anti-Leishmania drug. Parasites were diluted in a complete medium to 5 × 106parasites/mL and 100μL of the suspension were seeded into the plates, incubated at 22 °C for 72 h and then 20μL of MTT solution (5 mg/mL) were added into each well for 3 h. The plates were then centrifuged, the supernatants discarded and the resulting pellets dissolved in 100μL of lysing buffer consisting of 20% (w/v) of a solution of SDS (sodium dodecyl sulfate, Sigma) and 40% of N, N-dimethylformamide (Merck) in H2O. The absorbance was measured spectrophotometrically at a test wavelength of 550 nm and a reference wavelength of 650 nm. The results are expressed as IC50which is the dose of Neem oil necessary to inhibit cell growth by 50%; each IC50 value is the mean ± SD of at least three separate experiments per-formed in duplicate.
2.7.1. In vitro intracellular amastigote susceptibility assays
THP-1 cells (human acute monocytic leukemia cell line) were main-tained in RPMI supplemented with 10% FBS (Fetal Bovine Serum, EuroClone), 50μM 2-mercaptoethanol, 20 mM Hepes and 2 mM gluta-mine at 37 °C in 5% CO2. THP-1 cells were plated at 5 × 105cells/mL in 16-chamber Lab-Tek culture slides (Nunc) and treated with 0.1μM phorbol myristate acetate (PMA, Sigma) for 48 h to achieve differentia-tion into macrophages. Cells were then washed and infected with metacyclic L. infantum or L. tropica promastigotes at a macrophage/ promastigote ratio of 1/10 for 24 h. Macrophages were then washed and incubated at 37 °C in the presence of different concentrations of Neem oil for 72 h. Miltefosine was used as the control drug. Slides werefixed with methanol and stained with Giemsa. The percentage of
infected macrophages in treated and non-treated cells was determined by light microscopy. The results are expressed as IC50, which is the dose of compound necessary to induce a 50% reduction of infection. 2.7.2. Cytotoxicity against differentiated THP-1 cells and selectivity index
THP-1 cells were plated at 5 × 105cells/mL in 96 wells
flat bottom microplates and treated with 0.1μM PMA for 48 h to achieve differenti-ation into macrophages. Cells were then treated for 72 h with serial dilutions of Neem oil and cell viability evaluated using the MTT assay. The results are expressed as IC50, which is the dose of compound neces-sary to inhibit cell growth by 50%.
2.8. Antifungal activity against Candida spp. and Malassezia furfur The antimicrobial activity of Neem oil was evaluated by a microbroth dilution method according to the National Committee for Clinical Labo-ratory Standards (NCCLS) for Candida spp. and modified for Malassezia furfur (NCCLS, 2008; Rojas et al., 2014). The Minimum Inhibitory Con-centration (MIC) was determined as the lowest conCon-centration of natural compound at which no fungal growth was observed. Twenty-six strains of Candida spp. including 15 Candida albicans (13 clinical isolates, ATCC 24433 and ATCC 10231) strains, 7 clinical isolates of C. glabrata, 2 clini-cal isolates of C. krusei and 2 of C. tropiclini-calis and 20 strains of M. furfur (19 clinical isolates and CBS 7019 strain) were considered in this study. Candida spp. strains were cultured in plates at 96 wells with RPMI 1640 and MOPS (3-(N-morpholino)propanesulfonic acid) at 28 °C for 24–48 h supplemented with Tween 80 (final concentration of 0.01% v/v). To support the growth of lipid dependent yeast, the
Table 2
Quali–quantitative multicomponent pattern of Neem oil for polyphenols.
Compound μg/mL⁎of Neem oil 3-OH-4-MeO-benzaldehyde 5.48 ± 0.95 Benzoic acid 12.6 ± 1.4 t-cinnamic acid 1.9 ± 0.2
Naringenin 3.97 ± 0.34
TOTAL 23.95 ± 1.23
⁎ Data reported are the mean values ± SD on three independent determinations.
Fig. 1. Reflectance curves of Neem oil compared to those of Hemp oil and Olive oil. The reflectance curve is a potential fingerprint of the color expression of a sample, therefore the comparison with other simple matrices allows a better understanding of the expressed values. The curve slopes, between 500–570 and 690–700 nm, simply account for the different luminance, so that the Neem oil curve acquires an intermediate position. Values recorded around 590 nm show the yellow color region, while those recorded around 670 nm corresponds to the red color, so that the Olive and the Neem oil curves cross each other twice, with the increase and decrease of their a* and b* values. The positive yellow b* value of Neem oil, lower if compared with Olive oil, could account for a discrete content of polyphenols andflavonoids as further reported, while the higher positive a* value could account for a red component, not identified to date. This preliminary characterization could be useful for next studies on Neem oil composition, with particular attention to, potentially active red components, whose color could be correlated with the antimicrobial activity. These compounds could account for the color differences between this and other kinds of oils, such as hemp and olive oils, and the colorimetric parameters could be used to predict a biological activity.
Table 1
Colorimetric dataaof Neem, Hemp and Olive oil: A comparison.
Neem Hemp Olive
L* 46.1 ± 0.4 37.8 ± 0.2 50.0 ± 0.8 a* 12.2 ± 0.2 5.40 ± 0.04 4.6 ± 0.3 b* 35.7 ± 0.8 21.9 ± 0.2 42.5 ± 1.4 C*ab 37.8 ± 0.8 22.6 ± 0.2 42.7 ± 1.4 hab 71.1 ± 0.1 76.16 ± 0.07 83.8 ± 0.3 a
RPMI1640 medium was supplemented with glucose (1.8%), peptone (1%), ox bile (0.5%), malt extract (0.5%), Tween 20 (0.5%), Tween 80 (0.05%), glycerol (1%) The inoculum size was 2.5–5 × 103cell/mL. The plates were incubated at 32 °C and visual reading was performed after 24 h for approximately 3 days. The dilutions of Neem oil ranged from 12,500 to 12μg/mL.
3. Results and discussion 3.1. Color analysis
The tristimulus colorimetry was employed in this study to evaluate the color properties of A. indica oil. Some data are available in literature (Kumar and Parmar, 1996) about colorimetric studies on many different Neem oil samples. The authors described a large variety of colors (pale yellow, yellow, olive yellow, light olive brown, dark reddish brown and strong brown), but the only reported data concerned the hue rang-ing are between 2.5 Y and 10.0 R, in the orange-red zone of color. In this work, applying the described measure conditions, a significant yellow parameter (+ b*) can be shown apart from a weak red value (+ a*) that, together with the quite bright L* value, account for the light red-dish brown characterizing Neem oil. This could be due to the combina-tion of the phenolic components, with some red component rather than to a complex mixture of chemicals whose complexity provides a light brown nuance. The colorimetric data and the reflectance graph were re-ported inTable 1andFig. 1, where they are compared with those ob-tained from samples of Hemp and Olive oils.
3.2. Phenolic pattern by HPLC-PDA
Other authors studied the total phenolic (104.56 mg GAE/g dry weight extract) andflavonoid (26.91 mg CE/g dry weight extract) con-tent of seeds hydroalcoholic extract (Naseer et al., 2014). Specifically,
Singh et al. (2005)reported the quantification of four selected phenolic acids (tannic, gallic, chlorogenic and ferulic) after methanol extraction of the seeds and studied their presence and amount during the ripening process. According to our HPLC-PDA validated procedure, these pheno-lic acids were not detected in pure Neem oil whereas benzoic acid (12.6 ± 1.35μg/mL) and t-cinnamic acid (1.9 ± 0.2 μg/mL) were the most abundant (Table 2). Among the other phenols,
3-OH-4-MeO-benzaldehyde was also detected (5.48 ± 0.95μg/mL). Interestingly, we found a discrete amount (3.97 ± 0.34μg/mL) of naringenin. To the best of knowledge, these metabolites were evidenced in Neem oil for thefirst time. The remaining secondary metabolites of our validated HPLC procedure were not detected or were below Limits of Quanti fica-tion or Detecfica-tion.
3.3. Enzyme inhibition assays
The inhibitory potential of Neem oil was evaluated against cholines-terases, tyrosinase,α-amylase, α-glucosidase and lipase. The results are summarized inTable 3. The oil had an inhibitory effect on AChE (1.43 mg GALE/g oil) but it was not active on BChE. Similarfindings were also found by several researchers, who reported different results for cholinesterases (Murray et al., 2013).Vinutha et al. (2007)reported that the water extract from stem bark of A. indica exhibited a low inhib-itory effect on AChE (5.89% at 100μg/mL). Tyrosinase is a main enzyme in the melanin synthesis and the inhibition of this enzyme is therefore a useful way to control hyperpigmentation problems. From this view-point, Neem oil exhibited significant tyrosinase inhibitory potential with 59.36 mg KAE/g oil. The observed results could be linked to the chemical composition of this oil, which is rich in terms of benzoic acid. Indeed, benzoic acid and its derivatives were reported as good tyrosi-nase inhibitors (Khan et al., 2010), as benzoic acid can chelate the cop-per in the active site of this enzyme as one of its inhibition mechanisms (Chang, 2009). Therefore, Neem oil might be considered as a source of natural tyrosinase inhibitors. In accordance with ourfinding,Manosroi et al. (2014)previously stated that A. indica var. siamensis extracts and its isolated compounds (limonoids) had great potential in terms of inhibiting melanogenesis.
Regardingα-amylase and α-glucosidase inhibitory effects, these po-tencies were found to be 1.58 and 6.27 mmol ACAE/g oil, respectively. From these results, Neem oil may be a promising source for preparing novel antidiabetic products. Ourfindings are in line with previous stud-ies (Mukherje and Sengupta, 2013). For example, an A. indica extract has been patented asα-glucosidase inhibitor bySengupta and Mukherje (2008). Moreover, the plant or their constituents were reported as hy-poglycemic agents (Mukherje and Sengupta, 2013; Khosla et al., 2000). As expected, Neem oil had also a strong inhibitory effect on lipase with 101.29 mg OE/g oil and this concurs well withDechakhamphu and Wongchum (2015), who reported that the leaf extract of A. indica showed a 34.85% inhibition against pancreatic lipase. The observed
Table 5
In vitro antimalarial activity against D10 (CQ-sensitive) and W2 (CQ-resistant) strains of P. falciparum. D10a W2a IC50(μg/mL) IC50(μg/mL) Neem oil 198 ± 58 241 ± 7 Chloroquine 0.008 ± 0.001 0.19 ± 0.06 a
The results are the mean ± SD of IC50of three different experiments performed in
duplicate.
Table 4
MIC and MBC (μg/mL) values of Neem oil against eight H. pylori strains.
H. pylori strains MIC/MBC of Neem oil Antibiotics susceptibility MIC (μg/mL)
190 128/128 MNZs; CLRs; AMXs MNZ 1; CLR 0.064; AMX 0.032 23 128/128 MNZs ; CLRs ; AMXs MNZ 1; CLR 0.064; AMX 0.032 110 R 128/128 MNZr ; CLRs ; AMXs MNZ 128; CLR 0.03; AMX 0.032 NCTC 11637 128/128 MNZr ; CLRs ; AMXs MNZ 256; CLR 0.064; AMX 0.032 F1 128/128 MNZs ; CLRr ; AMXs MNZ 2; CLR 4; AMX 0.064 F40/499 64/64 MNZr ; CLRr ; AMXs MNZ 32; CLR 8; AMX 0.016 F4 128/128 MNZr ; CLRr ; AMXs MNZ 32; CLRN256; AMX 0.064 E17 128/128 MNZs; CLRrAMXs MNZ 2; CLRN256; AMX 0.064
MNZr = metronidazole-resistant; MNZs = metronidazole-susceptible; CLRr = claritromycin-resistant; CLRs = claritromycin-susceptible; AMXs = amoxicillin-susceptible. Table 3
Enzyme inhibitory effects of Neem oil.
Enzyme inhibitory assays Results⁎
AChE inhibition (mg GALAE/g oil) 1.4 ± 0.1 BChE inhibition (mg GALAE/g oil) na Tyrosinase inhibition (mg KAE/g oil) 59.4 ± 3.2 α-Amylase inhibition (mmol ACAE/g oil) 1.6 ± 0.2 α-Glucosidase inhibition (mmol ACAE/g oil) 6.3 ± 0.7 Lipase inhibition (mg OE/g oil) 101 ± 2 ⁎ Values are expressed are means ± S.D. of three parallel measurements. GALAE: Gal-antamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; OE: Orlistat equivalent; na: not active.
antilipase activity may be linked to the presence of benzoic acid, as it was identified byKaramać and Amarowicz (1996)as one of the stron-gest lipase inhibitors among several phenolic acids. The evidence from this study suggests that Neem oil could be considered as an alternative source of natural agents to combat global health problems, including Alzheimer's disease, diabetes mellitus and obesity.
3.4. Anti-Helicobacter pylori activity
The inhibitory activity of Neem oil against eight H. pylori strains is shown inTable 4. All strains, regardless of their antibiotic susceptibility, showed a MIC value of 128μg/mL except one displaying a MIC value of 64μg/mL. Following the indications ofKuete (2010), Neem oil could be considered as a moderate natural inhibitor but close to having signi fi-cant activity because both antibacterial (in term of MIC50) and bacteri-cidal (in term of MBC90) activity occur at the same concentration of 128μg/mL effects. As it is normal that more or less 1 log2 dilution step (minor error) can occur when the microdilution method is performed in different days, we are confident that our results could be considered to have a significant activity against Helicobacter pylori.
3.5. Antimalarial and anti-leishmanial activity
Neem oil was tested against W2, chloroquine (CQ)-resistant and D10, chloroquine-sensitive strains of P. falciparum. The data inTable 5
indicated that Neem oil possesses comparable inhibitory activity against both strains, suggesting that there is no cross-resistance to chloroquine. However, the activity was low and the IC50s were higher than those observed by other authors using leaf and seed extracts or isolated com-ponents (MacKinnon et al., 1997; Dhar et al., 1998). These differences could be due to non-standardized methods used for the preparation of the extracts, or to the parts of the plant used containing different con-centrations of the active components such as nimbolide, gedunin and epoxyazadiradione.
Neem oil was also tested against the promastigote stage of L. infantum and L. tropica. As shown inTable 6, the IC50s against both species were more than 200μg/mL indicating that the activity against promastigotes were about 4–5 times lower than that observed against L. donovani promastigotes treated with ethyl acetate fraction of leaf ex-tracts (Dayakar et al., 2015). Neem oil also showed inhibitory effects against intracellular amastigotes with IC50of 15.3 and 17.6μg/mL against L. infantum and L. tropica, respectively (Table 6). Cytotoxicity evaluation against differentiated THP-1 resulted in low toxicity on human macrophages and high selectivity index which indicate a more potent activity against amastigotes than cytotoxicity against uninfected cells thus suggesting a very good level of selectivity towards Leishmania spp.
3.6. Antifungal activity
In a previous report, the inhibition of clinical isolates of C. albicans by Neem oil obtained by steam distillation was reported to be weak with respect tofluconazole, but there was no detailed description of the chemical composition or characteristics of the oil (Agarwal et al., 2010). No information was also available for the inhibitory activity
against Malassezia spp., despite the importance of natural products in the treatment of cutaneous Malassezia-related diseases (Angiolella et al., 2017).
The antifungal activity of Neem oil against Candida spp. and Malassezia furfur was expressed as the MIC. In this study the geometric mean (MICM) was reported. For all Candida spp., MICM values were N 12,500 μg/mL. Neem oil was not active against yeasts and the antifun-gal activity was not different among C. albicans and other non-albicans spp. The same result was reported for M. furfur strains. A threshold value for natural products has been defined as follows: MIC below 100μg/mL (significant activity), 100 ≤ MIC ≤ 625 μg/mL (moderate ac-tivity) and MICN 625 μg/mL (low activity) (Kuete, 2010). Thus, Neem oil was endowed with a low inhibitory activity against the growth of Candida and Malassezia spp. This is also in agreement with the mod-erate inhibitory activity against Aspergillusflavus (520 μg/mL) and A. parasiticus (595μg/mL) reported for the hydroalcoholic extracts of the seeds (Naseer et al., 2014).
4. Conclusion
Neem oil was characterized, beyond the limonoid content, in terms of the color characteristics and the phenolicfingerprint, showing the presence of a high content of benzoic acid. The great inhibitory efficacy against important enzymes, such as tyrosinase, correlated with the high content of benzoic acid, and lipase makes it an interesting matrix to be used in the prevention or care of hyperpigmentation problems and obesity.
Moreover, antimalarial and anti-leishmanial activity were reported in addition to its safety profile against THP-1 cell line. On the other hand, albeit tested, weak or no activity was evident against H. pylori, Candida spp. and Malassezia furfur strains. Overall, thesefindings on pure Neem oil open new scenarios for the recognition of this natural product for the treatment of different diseases and could rationally jus-tify the ethnobotanical uses in traditional medicine.
Conflict of interest
Authors declare nofinancial/commercial conflicts of interest. Acknowledgements
This work was supported by local grants from“G. d'Annunzio” University of Chieti-Pescara (FAR 2017).
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi. org/10.1016/j.sajb.2018.10.019.
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