Inhibitory effects of Azadirachta indica secondary metabolites formulated cosmetics on some infectious pathogens and oxidative stress radicals

The chemicals and reagents used included the following: Mueller Hinton agar from Oxford Ltd. (Hampshire, England), Dimethyl sulfoxide (DMSO) from Fluka Chemicals (Buchs, Switzerland). 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were bought from Sigma - Aldrich (St Louis, USA). Reagents used were all analytical grade. The industrial cosmetics chemicals (caustic sodium, sodium silica, glycerol, shea butter, sodium citrate, cetyl alcohol, stearic acid, vitamin E, and propylparaben were bought from industrial soap and cosmetics market Ojota, Lagos, Nigeria. The A. indica dried seed (500 g) was bought from National research institute for chemical technology, Zaria, Nigeria. The A. indica leaves (200 g) and stem-bark (200 g) were collected in July 2015 at the Digital Institute Bridge, Lagos, Nigeria. A plant taxonomist (Mr. T.K. Odewo) at the Department of Botany, University of Lagos authenticated the three samples (neem seed, leaf, and stem-bark). The three voucher specimens (reference numbers LUH 2009A, LUH2009B, and LUH2009C) were kept in the Lagos University herbarium (LUH). The neem seed oil (fixed oil) was extracted with Federal Institute of industrial research Lagos Nigeria mechanical cold press at room temperature (25.3 ± 2 °C). The EOs were extracted from the air-dried leaves and stem-bark as described in the previous report [14]. The yield of each extract was calculated per gram of the plant material and the EOs were kept in vials at 4 o C until further analysis.

The physicochemical properties of the neem oil (NMO) and neem soap were performed using guidelines of AOAC [15] and as described by Fatiany et al. [16]. (a) Acid value: The AV (mg/ KOH/g) of the NMO was evaluated by neutralizing the free acids by KOH; (b) Saponification value (SV): The SV (mg/ KOH/g) was calculated as the quantity of KOH necessary to saponify esters of acids and to neutralize the free acids in one gram of the NMO; (c) Ester index (EI): The EI was quantified as the difference between the SV and the AV. (d) Relative density (RD): The RD was measured with an Anton Paar GmbH dosimeter (DMA35N USA); (e) Refractive index (RI): The neem oil RI was measured at 38 °C with a digital refractometer (ATC95200–009 France). The physicochemical parameters (pH, moisture, total fatty matter, total free caustic alkali, unsaponifiable matter, matter insoluble in ethanol and water) of the neem soaps were carried out as described in Mak-Mensah et al. [17] report. The SM in the leaves EO (LEO) and stem-bark EO (SBEO) were analyzed by gas chromatography-mass spectrometry (GC/MS) and retention index (RI). The instrument used (Hewlett- Packed 5973 mass spectrometer linked with an HP 6890 gas chromatograph) was programmed as in our previous report [14]. Thereafter, the identification each SM was carried out by the conformity of their mass spectra data (MSD) with the reference held in the computer library (Wiley 275, New York). Furthermore, the retention index (RI) of the SM was matched with those in literature, while the total percentage composition of SM in each EO was their peak areas.

The neem soap (NMS) was produced using standard recipe Hui [18] for conventional toilet soap making with the neem seed oil substituted for palm kernel oil (PKO) which provides the triglyceride (fatty acid). The full boiled method was used to produce sample. Briefly, the weighed neem oil and shea butter (Table 1) were warmed at 60 °C. The oil was filtered and poured into a 500 L beaker. Caustic soda solution (10% sodium hydroxide and 20% water) was added to the oil, stirred with a dried wooden stirrer for proper mixing. The mixture was heated, boiled and allowed to become slurry on continuous stirring. On cooling (34 ^o C), the slurry was divided into four equal weights, sodium silicate added to each and stirred properly. To the first part (NMS) no neem essential oil was added (as control), in the second part the extracted neem leaves EO was added (NMLEO), for the third part EO from stem-bark was added (NMSBEO), and the fourth contained both NMLEO and NMSBEO (1,1). Each slurry was separately stirred for proper mixing and left in a separate wooden mound to solidify. Thereafter, they were cut into smaller sizes and left at ambient temperature for 48 h prior to analysis.

The neem cream was produced using varnishing base formulation by combining deionized water phase (DWP) and neem seed oil (NSO) as oil phase (OLP) with the use of the emulsifier as described by Hui [17]. Briefly, the OLP was prepared by combining all the waxes (Table 2) and NSO in a microwavable container and heat until they are melted at 82.2 °C. The DWP was made by heating water separately to 75 °C, thereafter citric acid, methylparaben, and propylparaben were dissolved. The mixture then added to the OLP and blended thoroughly. On cooling (32 o C) it was divided into four equal weights in sterilized beakers. Subsequently, the 4 different neem cream samples (NMC, NMCL, NMCSB, and NMCLSB + NMCSB) were produced in a similar model as described four soap samples. They were kept in sterilized bottles at ambient temperature prior to analysis.

Five reference bacterial strains and two laboratory strains from our laboratory stock culture confirmed to be multi-drug resistant bacteria [19, 20] were used for the antibacterial assay. The reference and laboratory strains bacteria are four Gram-positive: S. aureus (NCIB 50080), M. smegmatis (ATCC 700084), L. ivanovii, (ATCC 19119), S. uberis (ATCC700407) and three Gram-negative bacteria: E. cloacae (ATCC 13047), E. coli 180 and V. paraheamolyticues reported to be resistant to sulphamethoxazole, ampicillin, streptomycin, cefuroxime, cephalexin, tetracycline and nalidixic [21] were tested against the neem cosmetics (NC), following CLSI (2014) procedures. The bacterial suspensions were made by inoculating a fresh stock culture of the test bacteria strains into tubes containing 5 mL of sterile Luria Bertani broth and incubated for 24 h at 37 °C. Thereafter, active cultures were grown 24 h in sterile Luria- Bertani broth inoculated into Mueller-Hinton Agar (MHA) incubated for 24 h at 37 °C. After incubation, single colonies were transferred from MHA plates into 4 mL of normal saline solution determined spectrophotometrically at 580 nm as previously reported by Omoruyi et al. [22] and adapted by Igwaran et al. [23] and the dilutions matching with 0.5 Mc-Farland standards were used for the assay.

The modified method of Gullon et al. [24] was used for the determination of MIC and MBC of the neem cosmetics (NC). Under the aseptic condition, two-fold serial dilutions were carried in sterile microcentrifuge tubes in a total volume of 100 μL of Muller Hinton (MH) broth mixed with the NC of various concentrations ranging from 0.0125–0.800 mg/mL. The positive and negative controls were ciprofloxacin and water respectively, thereafter they were incubated at 37 °C for 24 h. The was assay carried in triplicate and tubes with the lowest concentration without visible growth were reported as the minimum inhibitory concentration (MIC). The minimum bactericidal concentration (MBC) for each neem product and the control were determined by streaking out all tubes without visible growth in the MIC technique into fresh nutrient agar plates. Then, at 37 °C, the culture was incubated for 24 h. The lowest concentration of the cosmetics and ciprofloxacin that didn’t on the solid medium yield any growth after 24 h [25] was recorded as the MBC.

Three antioxidant assays (DPPH, nitric oxide, and lipid peroxyl) were used to assess the radical scavenging property of neem cosmetics. The DPPH procedures of Liyana-Pathirana et al. [26] as described in the previous report [14] was followed to evaluate the neem cosmetics (NC) inhibitory potentials against DPPH radical in DMSO as diluent. The test was performed in triplicate and the capacity of NC to scavenge DPPH • to a neutral molecule (DPPH-H) was determined as % scavenging effects with the eq. (1) below.

Where Abs cl is the absorbance of the DPPH radical + DMSO; Abs sp. is the absorbance of DPPH radical + NC or reference compounds (RCs).

The antioxidant potency of each NC and reference compound against the lipid peroxyl radical (LP •) were determined by the thiobarbituric acid assay as described in the previous report [14] with a known lipid-rich source (egg yolk). Subsequently, at 532 nm the absorbance of the solution was read after aspirating the upper organic layer. The scavenging efficacy of each NC and RCs against lipid peroxyl at increasing concentrations (0.025–0.50 mg/mL) was assessed with the eq. (1) above.

The sodium nitroprusside nitric oxide radical (NO•) induced protocol as described by Makhija et al. [27] was used to determine the NO• scavenging potential of the NC. At physiological pH 7.2, sodium nitroprusside nitric oxide decomposed generating NO• which react in the presence of O2 and resultant stable nitrite and nitrate is measured in Griess solution. Briefly, 1.0 mL of the NC at double fold concentrations (0.025–0.5 mg/mL prepared in DMSO) was added to 1.0 mL (10 mM) of sodium nitroprusside solution. The solution was incubated for 110 min at ambient temperature. Then followed the color development by adding 1.0 mL of the aliquot to Griess solution (1.0% N-naphthyl-ethylene diamine hydrochloride in 2.0% orthophosphoric acid and 1.0%, sulphanilamide). Thereafter, the absorbance of the color developed was then measured spectrophotometrically at 546 nm against the reagent blank. The scavenging effect (%) was then obtained using equation (a) described in DPPH radical test. The assay was carried out in parallel triplicate and the mean value calculated. The IC50 (mg/ mL) was obtained from the standard curve for each neem product and positive control in the three protocols. The lower the IC50 value the higher the antioxidant capacity.

The in vitro hemolytic technique as illustrated by Helander et al. [28] was followed to assess the cytotoxicity of the NC with few modifications. Hemolytic test investigates hemoglobin discharge in the plasma (indicating red blood cell [RBC] lysis) due to contact with the test sample. Human RBC from one of our projects involving human blood was used in preparing blood agar plates (BAP). Wells were bored on the BAP at 0.10–0.70 mg / mL prepared in deionized water. Then, into each well 25 μL of the neem product was poured while BAP and well with only deionized water was used negative control. Thereafter, at 37 °C, the plates incubated for 24 h. The presence of hemolytic activity was examined on wells and the assay was carried in triplicate.

The inhibitory effects (supporting document 1) of the neem cosmetics produced with and without AISM on test pathogens are as presented in Tables 3 and 4. The antibacterial activities of the neem soaps produced with AISM (NMLS, NMSBS, and NMLSBS) were significantly different (p < 0.05) from those without AISM (NMS) with MIC values ranging between 0.0125 to 0.400 mg/mL and 0.400 to 0.800 mg/mL respectively against the seven test pathogens. The NMS demonstrated a bacteriostatic effect on all test bacterial strains at 0.800 mg/mL, while the neem soap produced with AISM from the leaf EO (NMLS) was bactericidal against E. cloacae, L. ivanovii, S. aureus, and V. paraheamolyticues at 0.400, 0.400, 0.800 and 0.800 mg/mL respectively. Unlike the NMS and NMLS, the neem soap made with AISM from the stem-bark EO (NMSBS) was bactericidal against all the test pathogens with MBC ranging between 0.100–0.800 mg/mL. The neem soap prepared using AISMs from both leaf and stem-bark EOs (NMLSBS) exhibited the highest antibacterial effects with MIC and MBC values of 0.0125 and 0.025 mg/mL against L. ivanovii, and 0.025 and 0.200 mg/mL against E. cloacae. Interestingly the NMLSBS and positive control (ciprofloxacin) had same MIC value against L. ivanovii, while the MBC (0.025 mg/mL) was significantly better than control drug [ciprofloxacin (0.05 mg/mL)] as shown in Table 3.

The four neem creams were generally less active against the seven pathogens compared to the neem soaps. Like the neem soaps, the neem creams produced with the AISM stem-bark and from both leaf and stem-bark EO (NMLSBC) had better effects against the pathogens (Table 4). However, at 0.800 mg/mL on E. cloacae, the four creams exhibited a similar effect, while NMLSBC displayed a superior MIC value (0.400 mg/mL). The NMLSBC displayed higher inhibitory effects than other neem cream products with lower MBC values against the five test pathogens, however, its effect was lower than that of positive control drug (Table 4).

The effects of AISM from leaf and stem-bark EOs on the radical scavenging property of neem soaps (NMLS, NSBS, and NMLSBS), creams (NMC, NMLC, NMSBC and NMLSBC) as well as neem cosmetics (NMS and NMC) produced devoid of AISM were examined on three different radicals (DPPH •, LP •, and NO •). The radical scavenging effects of neem soaps and positive control (ascorbic acid and rutin) on the radicals were concentration dependent (Figs. 1, 2 and 3). At low concentration, Table 5 (0.10 mg/ mL) the neem soaps except for NMSBS scavenging effects on the three (DPPH, LP, and NO) radicals were significantly different # (p < 0.05), while the effect of NMSBS against LP •, and NO • were similar (s). At a higher dose (0.20 mg/ mL) all test neem soap samples scavenged the three radicals differently (Fig. 2). Similarly, at the highest concentration (0.5 mg/mL) the neem soaps excluding the soap without AISM scavenging effects were significantly different # (< 0.05) as showed in Fig. 3.

A substance that offers one or more hydrogen atoms to a radical converting the latter a non- radical molecule is referred to as a radical scavenger. This activity is shown as the color of DPPH • turns to yellow from purple (DPPH • solution) of the investigated substance because of formation of a neutral molecule of DPPH-H upon donation of H atom from the substance [29]. The strength of the examined radical scavenger is the reduction of UV absorbance at 517 nm. However, DPPH technique doesn’t distinguish radical type but an overall radical scavenging potency of a substance [14]. We therefore quantitatively tested presumed and précised inhibitory efficiency of each neem product by employing two different specific types of radicals (nitric oxide and lipid peroxyl) associated with skin inflammation, blood diseases, and other oxidative stress-related ailments. Evaluating the IC₅₀ values for the different soaps and creams, regression equations from the standard curves generated for each radical was used while t-test analysis was employed for significantly different of % radical scavenging effects against the concentrations (Figs. 1, 2 and 3). The neem cosmetics made without the AISM exhibited mild antioxidant capacity against nitric oxide, lipid peroxyl and DPPH radicals with IC50 values ranging between 4.78–6.50 and 6.10–10.42 mg/mL for the soaps and creams respectively. The cosmetics produced using AISM from the neem leaf and stem-bark EOs demonstrated the highest antioxidant efficacy against three distinct radicals with IC₅₀ values 1.65–2.60 mg/mL for the neem soap (NMLSBS). Interestingly, the antioxidant capacities of NMLSBS and NMLSBC were remarkably better than the two positive control drugs (Table 6) against LP and NO radicals associated with OSD.

The physicochemical properties (PCP) of the crude neem seed oil (NSO) and essential oils (EOs) extracted by mechanical cold press and hydrodistillation from the aerial parts respectively are as presented in Table 7. Except a lower iodine value (IV) of 77.47% obtained, the saponification, refractive index, and acid values of the NSO were similar to previous reports of neem seed oil [17, 30, 31] and some vegetable oils used for cosmetics making, especially soap [32]. However, the IV in this present study was higher than the result obtained (63.81%) for neem seed oil grown in Sudan [31, 33]. From Table 7, it can be noticed that the relative density (RD) of the NSO and the two EOs is less than 1. However, the RD of the crude NSO (0.921) was significantly lower (p < 0.05) than that reported for neem seed oil (0.939) by Akpan, [34] and higher (0.905) than that obtained in India [35]. The RD values for EO from leaves (0.0902) and stem-bark (0.904) was significantly different p < 0.05 from the crude NSO. The acid values of the EOs were found to be greater than NSO. Conversely, the ester, refractive indices and saponification values of the NSO were greater than that of leaf and stem-bark EOs.

The physicochemical properties of neem soaps produced with and without the AISM are as presented in Table 8. The chemical properties of neem soaps were within the control limits of South Africa specifications for antibacterial toilet soap, Kenya standard for commercial toilet soap and standard Organisation of Nigeria (SON) for toilet soap [36‐38], except the free caustic alkali (0.32% as Na₂O) for the neem soap (NMS) produced without AISM. Nevertheless, the total fatty matter (77.30–80.14%) for the four neem soaps and other physicochemical parameters were better than the specifications of the SON for toilet soap and similar to neem soap produced elsewhere [17].

The cytotoxicity effects on neem cosmetics are as presented in Table 9. DMSO in blood agar without the cosmetics was used as negative control. The neem soap produced using AISM from both the leaf and stem – bark (NMLSBS) exhibited low toxic (slight lysis) effect at 0.350 mg/ mL, while none of the cosmetics displayed acute toxic effect at less than 0.70 mg/mL on human RBC except NMLSBS.

The chemical profiles of secondary metabolites (SM) isolated from A. indica leaf and stem-bark incorporated unto the recipes of neem cosmetics are as presented in Table 10. In this current study terpenes and oxygenated terpenes (terpenoids) that were the chief SM, efficiently boosted the antibacterial and antioxidant properties of neem cosmetics. Fifteen SMs were found in the LEO, while the SBEO contained 19 which represents 98.91 and 99.45% of the total SM content respectively (Table 10). In the LEO, α-caryophyllene, γ-elemene and hexadecane were the major sesquiterpenes, accounting for 44.30% of the overall SM content, while the total monoterpenes, monoterpenoids and sesquiterpenoids SM were 22.70, 10.50 and 4.51% respectively. Conversely, in the SBEO there were fewer sesquiterpenes SM (24.21%) compared to the SM in the LEO. However, there were more sesquiterpenoids (23.70%) and monoterpenoids (19.80%) SM in SBEO. The presence of bioactive SMs including a diterpenoid (phytol 9.21%), curcumene (7.8%), camphene (3.0%), ledol (2.4%) and tetracosane (1.32%) in SBEO absent in LEO remarkably distinguished the chemical profile of the stem-bark EO from the leaf (Table 10). Furthermore, the yields of linalool oxide (15.2%), terpinene (13.7%), hexadecenoic acid (10.2%) and polyunsaturated linoleic acid (5.40%) were significantly higher in SBEO. This may corroborate the superior bioactive and physiochemical properties observed in neem stem-bark cosmetics products (NMSBEOS, NMLEOS-NMSBEOS, NMSBEOC, NMLEOC-NMSBEOC) in this study.