Nanoemulsion formulation process optimization
The oil-in-water nanoemulsion was formulated using suitable inert ingredients through low-energy emulsification approach. It is a spontaneous, simple and cost-effective process of formulating oil into nanoemulsion 18. This method can be accomplished by the aqueous phase or oil phase titration process. The NE (O/W) was prepared using neem oil, lemongrass oil, surfactants, botanical adjuvant, anti-freezing agent and water (Fig. 7). The concentration of the oil phase and surfactants were varied during the preparation and ternary phase diagram based on three components: surfactant, water and oil were generated 19. Atlox 4916 and Nonylphenol ethoxylates were selected for being non-ionic surfactants and hence least affected by pH and ionic strength 20. The 1:5 ratio of the NE was found to be stable with droplet size (25-50nm) and polydispersity index (<0.301) (Fig. 1) which is indicative of the system 21. Selection of optimal adjuvant blend is a key to stable nanoemulsion due to strong repulsive force that averts flocculation and coalescence between the nanodroplets 22. Lemongrass oil (Cymbopogon citratus) and Prosopis juliflora extracts were found to be suitable adjuvant for the formulation of stable NE since these are less affected by pH, and are considered to be nontoxic and biocompatible23. The stability of the NE can persist over longer period of time due to the presence of the stabilizing adjuvant that inhibits the coalescence of the nanodroplets.24,25
Effect of lemongrass oil on kinetic stability
Stability is one of the most important parameters in nanoemulsion system because of their small droplet size and large surface area. The small droplet size of nanoemulsions provides stability against sedimentation or creaming because of the Brownian motion and consequently the diffusion rate are higher than the sedimentation rate induced by the gravity force. A stability test of nanoemulsions was conducted by varying the storage time (0-14days) or temperatures (4-45°C). The study was accomplished by observing the sample appearance or measuring their physicochemical properties such as zeta potential or particle size at predetermined interval time and the samples without any major changes in their appearance likes phase separation, creaming, flocculation, coalescence and sedimentation was considered as a stable system.
In the present study, no phase separation, creaming or crystallization were observed in neem oil nano-emulsion with botanical adjuvant after storage for 14 days at low (0°C) and high temperature (45°C). The results indicate that neem nano-emulsion is kinetically stable formulation during storage for extended period of time23. Generally nanoemulsion formation requires external energy to increase Gibbs free energy (ΔG) in nano droplets for extended stability. Suitable adjuvant system is also necessary for improved kinetic stability of nano-emulsion. Lemongrass oil has been previously reported as stabilizer and co-surfactant for neem oil microemulsion20.
Role of Prosopis juliflora on droplet size, poly dispersive index (PDI) and zeta potential
Droplet size and zeta potential measurements are the general requirements to test the nano-emulsion stability24. The most common destabilization phenomenon like flocculation, creaming, and coalescence etc. are directly related to droplet size25, 26. The small size of nanoemulsion is desirable in achieving optimal efficiency. A complete picture of droplet size population and distribution in Neem NE formulations were obtained by analysis of data generated by dynamic-light-scattering (DLS). The average droplet size was found in the range of 25-50nm without any major changes in droplet size during storage. Nano-emulsions with smaller the droplet size will increase the Brownian motion and weaken the attractive forces in nano-emulsion systems to produce stable formulation27. During storage conditions, smaller droplets generally converted into larger droplet size by Ostwald ripening process which can be inhibited by selective surface active agents or adjuvant. Droplet growth rate is explained by the Lifshitz-Slezov and Wagner equation:-
In neem nano-emulsion, average droplet radius (r) during storage time (t) are reduced due to low interfacial tension (ϒ) generated by surface active agents or adjuvant and stabilizers which ultimately reduced the molecular volume of neem oil (Vm) at absolute temperature (T). Occurrence of Ostwald ripening was avoided by increasing the elasticity of droplet103 and the addition of natural adjuvant which reduced interfacial free energy forming a mechanical barrier against coalescence. Prosopis juliflora, may be acted as bio-polymeric adjuvant due to presence of cellulose fiber 28, 29 in this formulation.
Polydispersity values below 0.2 indicate uniformity among oil droplet sizes or monomodal distributions and therefore better stability, whereas values close to 1 indicate a heterogeneous or multimodal distribution 29. In the present study, values were closer to 0.3 indicating mono-dispersive nature of developed nano-emulsion with good stability during storage. The selection of PDI value which is less than 0.5 is acceptable for agricultural use and is considered as a good uniformity of the droplet diameter.
Zeta potential values provide the information about the homogenous behavior of the nano-emulsion. Particles with zeta potential more positive than + 30 mV or more negative than - 30 mV are usually considered to be stable, since electrical charge of droplets is strong enough to assume that repulsive forces between droplets are predominant in the nanoemulsion60.The surface of NE was negatively charged with an average zeta potential (−30 and −40mV) which indicates that the formulation is stable. A negative zeta potential value induces repulsive forces that are greater than the attraction forces among droplets, thus averting the coagulation and coalescence to occur in disperse emulsion. Zeta potential quantifies surface charge over the emulsion droplet indicating the physical stability of nano-formulations30. Zeta potential gives the electrophoresis mobility data related to the stability of NE formulation. Higher value indicates good stability of nano-formulation due to higher repulsion between the droplets to prevent coagulation and flocculation during storage31. Droplet size, PDI and zeta potential signify clear, uniform and homogenous micelle based neem nanoemulsion formulation with long term stability.
Botanical adjuvant on acidity/alkalinity (pH)
The surface properties around the droplet determine the pH value as an indicator of nanoemulsion stability. The pH of the formulations was found in the slightly acidic range (5.9-6.5). However, the formulations become almost neutral at application concentration (0.1–1%). Previous studies have reported that high alkalinity or acidity of the nano-formulations leads to degradation of neem ingredient which in turn, reduces the bio-efficacy of the formulation32. In another study it was reported that pH aggregates and destabilize the nano-emulsion during storage33. In the present study, it was observed that botanical adjuvant stabilizes the pH to slightly acidic (5.9-6.5) to reduce degradation (1.42%) of neem during storage at low, ambient and high temperatures.
Viscosity
Neem NE without botanical adjuvant was found to be less viscous (88.7cPs) compared to Neem NE with botanical adjuvant (98.8cPs). Optimum viscosity of a formulation is required for long term storage without sedimentation, complete transfer from container to spray tank, homogeneous dispersion for spray solution and adhesion and reduction of run-off from target surfaces34.Optimum viscosity also reduces the rate of aggregation in nano-emulsion during storage35. The viscosity value may be affected by the nature of surfactants, organic phase components and oil viscosity. Pesticide nanoemulsion produces low viscosity as it is categorized as O/W type with high water loading. However, the viscosity of nanoemulsion can be altered by surfactant concentration35. Results showed that Neem NE with botanical adjuvant can give long term stability during storage and improved adhesion on applied surfaces for good bio-efficacy.
Surface tension
Low surface tension (23.4mNm-1) in neem NE (with adjuvant) provides better spreading of formulation on brinjal leaves with good bio-efficacy (91.24%) compared to neem NE (without adjuvant) (33.6 mNm-1). Finer droplet size allows the spreading of droplet uniformly on the plant leaf surface36. Lower surface tension improves wetting, spreading and penetration into the applied surface of plants37. Wetting is the important parameter which directly linked with the contact angle and surface tension. Retention and contact angle of leaves are measured to relate the affinity of the pesticide liquid towards the leaf surfaces. Contact angle is the quantitative measurement of surface adhesion of liquid on solid surface38. Contact angle measurement is linked to Young’s equation;
SA = SL + LA cosθ-------------------------------------------------------(2)
Where:
SA= Surface tension between solid and air
SL= Surface tension between soilid and liquid
LA= Surface tension between liquid and air
Cos ϴ = Contact angle
The efficiency of neem nanoemulsion was enhanced by increasing the adhesion work of nanoemulsion towards the leaves. Neem nano-emulsion reduces the surface tension and contact angle due to spreading of oil droplets over leaf surface (Fig. 2). It worth noted that the contact angle of nanoemulsion decreased as the increasing P.juliflora content, showing that neem oil has low interfacial tension which effectively allowing neem oil diffusion in the plant surface104. Adjuvant along with water based neem nano-emulsion binds firmly over the leaf surface with grip induced by sticky adjuvant (Fig. 3). Neem nanoemulsion with P.juliflora conferred further lowering of surface tension may be due to the presence of cellulose polymer. So, the addition of botanical adjuvant improves wetting and dispersion of NE.
Effect of botanical adjuvant on active ingredient (azadirachtin) compatibility
FT-IR spectroscopy is a valuable technique for the identification of any functional group among various bioactive constituents39. FTIR spectroscopy of Neem nanoemulsion, neem oil and adjuvant was done and the obtained data were compared to identify the possible interaction of various functional groups that were involved in the synthesis (Fig. 4). Natural adjuvant P.juliflora showed broadband at 3314.96cm-1 due to hydroxyl groups of phenolic constituents, band at 1613cm-1 due to aromatic C=C and similar peaks have been previously reported by Kumara et.al. The study explained that functional groups were corresponds to flavonoid compounds40. In view of previous studies, hydroxyl and aromatic peaks may be due to the flavounoid constituents. Phenolic and alkene derivatives in P. juliflora extract may enhance the storage stability of azadirachtin 41.
Neem nano-emulsion shows the corresponding peaks of neem oil, azadirachtin constituent corresponding peaks at 1745.72 cm-1 and at 2848.17 cm-1 without any chemical modification. FT-IR data results concluded that azadirachtin active constituents were stable and compatible with botanical adjuvant in nano-emulsion formulation system.
Effect of natural adjuvant on active ingredient (azadirachtin) storage stability
In water-based formulations, azadirachtin content is generally degraded by hydrolysis reactions (Fig. 6) during storage to reduce the bio-efficacy. So suitable stabilizer is required to protect the azadirachtin from undesirable degradation. Analytical chromatogram of neem NE formulation obtained by HPLC analysis revealed the degradation pattern of azadirachtin (Fig. 5). Azadirachtin showed linear detector response (Fig. 8) over the concentration range (0.05-1.0mgL-1) tested, with correlation coefficient linear functions > 0.999 and regression equations, y = 93322x - 51.38. In the present study, P. juliflora acts as a stabilizing agent in neem NE formulation as results indicate that azadirachtin content remained stable with negligible degradation (1.42%) compared to NE formulation without P. juliflora (15.26%). This decrease might be due to the presence of glycosidic and phenolics compound in botanical adjuvant.
Half-life (t½) of azadirachtin in neem oil with and without of botanicals was observed and it ranged from 45.84 to 492.95 days (Table 1). The lowest t½ value was obtained without botanicals and increased manifolds with the addition of botanical synergist. An increase in the half-life of azadirachtin again signifies its stability due to the presence of botanical adjuvant.
Table 1: Degradation dynamics of azadirachtin in neem NE formulations during storage.
Duration of storage (Days)
|
Degradation of azadirachtin
|
Neem NE without adjuvant
|
Neem NE with adjuvant
|
Mean Degradation %
+
Standard Deviation
|
Mean Degradation %
+
Standard Deviation
|
1
|
-
|
-
|
4
|
2.0 + 0.1
|
0.07 + 0.01
|
10
|
7.1 + 0.1
|
0.09 + 0.01
|
14
|
15.26 + 0.15
|
1.42 + 0.07
|
Half–life (Days)
|
45.84
|
492.95
|
Azadirachtin is the secondary metabolite of neem, but high rate of degradation confines its efficient usage in controlling insects42. Farmers are not benefitted from neem products due to its low stability as reported in various research findings15. Some stabilizers have been used to reduce degradation of azadirachtin45, 46. All these stabilizers are good and efficient but due to their chemical nature and limited availability are less popular in farmers. Whereas, in the present study, naturally occurring botanical adjuvant was used to enhance the stability of azadirachtin in neem oil which is quite safe for plants as well as for human health also.
In-vitro insecticidal activity of Neem nanoemulsion against whitefly
The results of in-vitro bioassay of the selected formulations against whitefly were presented in Table 2. Mortality (%) of whitefly was increased significantly (p <0.05) with the increment of dose and time of exposure. Neem 20 NE (with adjuvant) exhibited the best insecticidal activity against whitefly with LC50 value of 2.05 mLL-1. Moreover, Neem 20 NE showed good mortality (56.1%) of target pest after 5days of treatment at 8mLL-1(Table 2) which was quite better than crude neem oil at 1% dose (Pissinati and Ventura, 2015). Neem 20NE formulation (with adjuvant) demonstrating the strongest insecticidal activity with the lowest LC50 value of 2.05mLL-1whcih indicates that Prosopis juliflora extract acted as a promising bio-efficacy enhancer in neem nano emulsion.
Table 2. In vitro insecticidal activity of neem NEs against whitefly.
Insecticide Formulation
|
Treatment/ Doses
|
Mortality (%) at hours of application
|
Lethal Concentration (mLL-1)
|
24hrs
|
48 hrs
|
72hrs
|
96hrs
|
LC50
|
LC90
|
Neem 20NE
|
1 mLL-1 (T1)
|
6.12
|
12.78
|
16.67
|
18.88
|
4.3
|
56.4
|
2 mLL-1 (T2)
|
18.88
|
30.55
|
38.33
|
41.12
|
4 mLL-1 (T3)
|
28.88
|
37.78
|
47.22
|
55.00
|
8 mLL-1 (T4)
|
36.67
|
42.22
|
48.88
|
56.12
|
Neem 20 NE+adjuvant
|
1 mLL-1 (T5)
|
12.78
|
20.55
|
23.33
|
26.67
|
2.05
|
12.33
|
2 mLL-1 (T6)
|
25.55
|
37.78
|
50.55
|
57.78
|
4 mLL-1 (T7)
|
34.45
|
47.78
|
56.12
|
63.33
|
1 mLL-1 (T8)
|
54.45
|
63.88
|
65.55
|
84.45
|
Cypermethrin
(10% EC)
|
1 mLL-1 (T9)
|
72.78
|
82.22
|
96.12
|
98.33
|
Control (Water)
|
1 mLL-1 (T10)
|
0.55
|
2.22
|
4.45
|
3.88
|
S.Em±
|
-
|
2.63
|
2.15
|
2.15
|
1.58
|
CD (p=0.05)
|
|
7.82
|
6.4
|
6.39
|
4.68
|
CV %
|
-
|
14.98
|
10.17
|
8.93
|
5.99
|
In-vivo insecticidal activity enhancement of neem NE against whitefly on brinjal
Neem oil has been widely reported as potent bio-pesticide due to anti-feedant, repellent, and growth-inhibiting properties47. Neem oil comprises various azadirachtin analogs that offer various insecticidal properties against different agricultural as well as household insect pests48. Azadirachtin imparts chemical defense against pest attack for efficient pest management strategies without hampering non-target organisms. Neem oil in comparison to synthetic pesticides has been extensively studied for the control of whiteflies which showed equivalent control as synthetic pesticides49.
Nano-emulsion is a new formulation technology for improving the bio-efficacy of neem oil for long term pest control50.Biological activity of neem NE can be improved further by addition of adjuvant or other inert ingredients51. The effect of adjuvant on neem nano formulation was evaluated on whitefly population and percent reduction was compared with control replication wise to nullify the impact of different factors in the field such as the presence of natural enemies, free movement of insects and conventionally used synthetic insecticide Cypermethrin 10% EC also (Table 3). Pre-treatment (PT) population of target pest was homogeneous before application and increased steadily over the period of observation till 14 days. Among formulations developed, Neem 20NE with adjuvant recorded significantly (p <0.05) low population of B. tabaci from 1 to 14 days after treatment @ 1000 mLha-1.
In the present study, botanical adjuvant was used as possible bio-efficacy inducer against whiteflies of brinjal. Neem 20 NE (without adjuvant) reduced population (43.30%) which was further enhanced to 91.24% by incorporating botanical adjuvant and equivalent to Cypermethrin 10% EC (95.23%). Inclusion of adjuvant (P. juliflora) efficiently increased the biological effectiveness of neem nano-emulsion. Insecticidal activity of n-hexane extract of P. Juliflora seed oil against termite (Odontotermes obesus) and cockroach (blattella germanica); antimicrobial activity of P. juliflora seed pods aqueous extract against some common pathogens 61 was reported earlier. Moreover, in presence of P. juliflora, neem oil nano-formulation showed possible synergistic activity (110.71%).
Table 3: Effect of botanical and synthetic formulations on whitefly infestation in brinjal crop
Insecticide Formulation
|
Treatment/ Doses (%)
|
Mean no. of whiteflies* per 3 leaves per plant at pre-treatment (PT) and different days (D) after treatments
|
% reduction of whitefly population±
|
PT
|
1D
|
3D
|
5D
|
7D
|
10D
|
14D
|
Neem 20 NE
|
1000 mLha-1 (T1)
|
1.10
(1.26)
|
1.06
(1.25)
|
1.01
(1.23)
|
0.91
(1.19)
|
0.86
(1.17)
|
1.27
(1.33)
|
1.47
(1.40)
|
43.30
|
Neem 20 NE with botanical adjuvant
|
1000 mLha-1 (T2)
|
1.06
(1.25)
|
0.33
(0.91)
|
0.21
(0.84)
|
0.18
(0.82)
|
0.15
(0.80)
|
0.12
(0.79)
|
0.23
(0.85)
|
91.24
|
Cypermethrin
(10 EC)
|
500 mLha-1 (T3)
|
0.98
(1.22)
|
0.40
(0.95)
|
0.29
(0.89)
|
0.27
(0.88)
|
0.07
(0.76)
|
0.06
(0.75)
|
0.12
(0.79)
|
95.23
|
Control
(Water)
|
500Lha-1
(T4)
|
1.14
(1.28)
|
1.23
(1.31)
|
1.26
(1.32)
|
1.52
(1.42)
|
1.95
(1.56)
|
2.28
(1.66)
|
2.59
(1.75)
|
0
|
S.Em±
|
-
|
0.05
|
0.06
|
0.06
|
0.06
|
0.05
|
0.05
|
0.05
|
-
|
CD (p=0.05)
|
-
|
0.15
(S)
|
0.16
|
0.17
|
0.16
|
0.15
|
0.15
|
0.16
|
-
|
CV %
|
-
|
6.94
|
8.09
|
8.31
|
8.21
|
7.38
|
7.65
|
7.53
|
-
|
*Values in parentheses are square-root transformed; PT: Pre-treatment; ± Compared to untreated (control) 14 days after application.
Tukey’s Honest Significant Difference (HSD) Test or Post-hoc analysis was performed to evaluate how Neem 20 NE (T1), Neem 20NE+adjuvant(T2), Cypermethrin 10% EC (T3) and untreated control (T4) treatments affect the behaviour of percent reduction of whiteflies at different days interval (Table 4). From this study, it was found that these four categories of treatments differ in the way they reacted towards whitefly population management. Test results indicated that Neem 20NE and Neem 20NE+adjuvant yielded significant differences (0.5203) on the reduction (%) of whiteflies. No significant difference accumulated for Cypermethrin (10% EC). It means that botanical adjuvant had strong influence on percent reduction of whiteflies.
Table 4: Tukey’s Honest Significant Difference (HSD) Test of botanical and synthetic formulation’s effects on whiteflies
Group (A)
|
Group (B)
|
Mean Difference
|
Std. Error
|
Sig.
|
95% Confidence Interval
|
Lower Bound
|
Upper Bound
|
Neem 20 NE
|
Neem 20 NE with botanical adjuvant
|
1.2400
|
0.05
|
0.5203
|
-3.7302
|
1.2502
|
Neem 20 NE
|
Cypermethrin
(10 EC)
|
-1.3500
|
0.06
|
0.0502
|
-4.1031
|
1.4031
|
Neem 20 NE
|
Control
(Water)
|
-1.1200
|
0.06
|
0.3903
|
-0.8089
|
3.0489
|
Neem 20 NE with botanical adjuvant
|
Cypermethrin
(10 EC)
|
-0.1100
|
0.06
|
0.0996
|
-3.2159
|
2.9959
|
Neem 20 NE with botanical adjuvant
|
Control
(Water)
|
-2.3600
|
0.05
|
0.0558
|
-0.0458
|
2.9959
|
Cypermethrin
(10 EC)
|
Control
(Water)
|
-2.4700
|
0.05
|
0.0773
|
-0.2069
|
5.1469
|
*The mean difference is significant at the 0.05 level.