Author’s Accepted Manuscript

Ultrasound-assisted extraction of azadirachtin from

dried entire fruits of Azadirachta indica A. Juss.
(Meliaceae) and its determination by a validated
HPLC-PDA method

Joelma Abadia Marciano de Paula, Lucas Ferreira

Brito, Karen Lorena Ferreira Neves Caetano,
Mariana Cristina de Morais Rodrigues, Leonardo www.elsevier.com/locate/talanta

Luiz Borges, Edemilson Cardoso da Conceição

PII: S0039-9140(15)30459-8
DOI: http://dx.doi.org/10.1016/j.talanta.2015.11.005
Reference: TAL16101
To appear in: Talanta
Received date: 16 June 2015
Revised date: 30 October 2015
Accepted date: 1 November 2015
Cite this article as: Joelma Abadia Marciano de Paula, Lucas Ferreira Brito,
Karen Lorena Ferreira Neves Caetano, Mariana Cristina de Morais Rodrigues,
Leonardo Luiz Borges and Edemilson Cardoso da Conceição, Ultrasound-
assisted extraction of azadirachtin from dried entire fruits of Azadirachta indica
A. Juss. (Meliaceae) and its determination by a validated HPLC-PDA method,
Talanta, http://dx.doi.org/10.1016/j.talanta.2015.11.005
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 Ultrasound-assisted extraction of azadirachtin from dried entire fruits of
Azadirachta indica A. Juss. (Meliaceae) and its determination by a validated HPLC-
PDA method

Joelma Abadia Marciano de Paula1,*, Lucas Ferreira Brito2, Karen Lorena Ferreira Neves
Caetano3, Mariana Cristina de Morais Rodrigues3, Leonardo Luiz Borges4, Edemilson
Cardoso da Conceição3

1
Unidade Universitária de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, Anápolis,
Goiás, Brazil
2
Curso de Farmácia, Instituto de Ciências da Saúde, Universidade Paulista, Goiânia, Goiás, Brazil
3
Laboratório de Pesquisa, Desenvolvimento and Inovação de Bioprodutos, Universidade Federal de Goiás,
Goiânia, Goiás, Brazil
4
Curso de Farmácia, Pontifícia Universidade Católica de Goiás, Goiânia, Goiás, Brazil

ABSTRACT

Azadirachta indica A. Juss., also known as neem, is a Meliaceae family tree from India. It
is globally known for the insecticidal properties of its limonoid tetranortriterpenoid
derivatives, such as azadirachtin. This work aimed to optimize the azadirachtin ultrasound-
assisted extraction (UAE) and validate the HPLC-PDA analytical method for the
measurement of this marker in neem dried fruit extracts. Box-Behnken design and
response surface methodology (RSM) were used to investigate the effect of process
variables on the UAE. Three independent variables, including ethanol concentration (%,
w/w), temperature (°C), and material-to-solvent ratio (g mL-1), were studied. The
azadirachtin content (µg mL-1), i.e., dependent variable, was quantified by the HPLC-PDA
analytical method. Isocratic reversed-phase chromatography was performed using
acetonitrile/water (40:60), a flow of 1.0 mL min-1, detection at 214 nm, and C18 column
(250 x 4.6 mm, 5 µm). The primary validation parameters were determined according to
ICH guidelines and Brazilian legislation. The results demonstrated that the optimal UAE
condition was obtained with ethanol concentration range of 75 – 80% (w/w), temperature
of 30°C, and material-to-solvent ratio of 0.55 g mL-1. The HPLC-PDA analytical method
proved to be simple, selective, linear, precise, accurate and robust. The experimental
values of azadirachtin content under optimal UAE conditions were in good agreement with
*
Corresponding author:
Joelma A. M. Paula
Campus Anápolis de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, Br 153, Nº310, Fazenda
Barreiro do Meio, Campus Henrique Santillo, Anápolis, Go, Brazil, Postal code: 75132-400
e-mail: joelma.paula@ueg.br, telephone number: 55 (62) 3328-1160
the RSM predicted values and were superior to the azadirachtin content of percolated
extract. Such findings suggest that UAE is a more efficient extractive process in addition to
being simple, fast, and inexpensive.

Keywords: Ultrasound-assisted extraction; HPLC-PDA; Box-Behnken design; Response

surface methodology; Azadirachtin; Azadirachta indica

1. Introduction

Azadirachta indica A. Juss (Meliaceae), also known as neem, is a native tree from
the Indian subcontinent long recognized for its particular insecticidal properties [1-4] and
its benefit for human health [5-7] and is thus considered a plant with multiple applications
in the medical, agricultural, and cosmetic fields, among others [8]. Neem is grown in most
tropical and subtropical world areas for reforestation and the production of raw material for
natural insecticides and medicines [9]. Neem’s primary active ingredient is azadirachtin
(Fig. 1), a limonoid tetranortriterpenoid found in nearly all parts of the plant, especially in
the seeds [6.9], with antifeedant, anti-growth, and ovicidal actions against a variety of
plague insects [8]. In addition to the azadirachtin, neem features a large variety of
triterpenoids, such as nimbin, salannin, azadirachtol, nimbidinin and gedunin [5]. Although
many isomers of azadirachtin, Aza-A to Aza-K, have been reported in the literature,
azadirachtin-A is the most important form and is used as a standard for expressing the
activity of neem extracts and their formulations [10].

[Insert Figure 1]

The immediate applications as an ecofriendly or biodegradable pesticide increase

the demand of raw materials from A. indica seeds rich in azadirachtin. However, practical
limitations are still observed, such as the handiness of seeds throughout the year, the
efficiency of extraction methods, and the availability of sensitive and accessible analytical
methods for use in quality control [11-13].
The seasonality in the production of neem seeds forces farmers to store the
harvested fruits for a certain period until commercialization. The neem fruiting starts after 3
to 5 years of planting, and the tree becomes completely reproductive after 10 years,
producing an average of 25 kg/tree/year in blooms that occur twice a year at most [14].
Usually, the mature fruits are collected from the ground after falling, dried in a ventilated
place, packed in bags that allow aeration, and kept in a cool and dry environment until
commercialization with industry sectors [15]. During the storage period, the dried fruit
azadirachtin levels may change due to degradation caused by storage conditions [16] and
should be monitored with the application of efficient and precise analytical methods.
The precision of the analytical methods applied to bioproducts depends on the
extraction method. Classical methods, such as maceration and percolation, are important
means of obtaining biologically active compounds from neem fruit [17]. However, to
shorten the extraction and obtain higher levels of the azadirachtin marker, several
methods are mentioned in the literature, such as mechanical pressing, supercritical fluid
extraction [11, 12], microwave assisted extraction [18] and pressurized hot solvent
extraction [19]. In this listing, we can add the ultrasound-assisted extraction (UAE). This
method’s extraction mechanism, assigned to the mechanical and cavitation forces caused
by sound waves, leads to the reduction of particle size, breakdown of the plant cell wall
and increased mass transfer through the membrane [20, 21], making it a cheap, simple,
fast and efficient alternative compared to conventional extraction techniques. This has
been seen in the recent application of UAE in the extraction of various plant compound
classes [21-26].
Several HPLC analytical methods have been proposed and used in the azadirachtin
quantification of A. indica extracts and byproducts [10, 11, 13, 16, 19, 27], but many of
them have been performed from complex extractive processes, making the sample
preparation step critical because it involves multiple phases, making the method more
difficult and expensive. Moreover, not all of the methods proposed in the literature were
validated for the parameters required by the official guidelines to ensure the selectivity,
linearity, precision, accuracy and robustness of the method.
This work aimed to optimize the azadirachtin ultrasound-assisted extraction and
validate the HPLC-PDA analytical method for the measurement of this marker to propose
a precise, simple and inexpensive method for monitoring the azadirachtin content of neem
dried fruits and its bioproducts.

2. Experimental

2.1 Chemicals and reagents

Ethanol 95% (v/v) (Vetec, analytical grade), methanol and acetonitrile (J.T. Baker;
HPLC grade), and water filtered through a Milli-Q apparatus (Millipore) were used in
sample and mobile phase preparations. Azadirachtin analytical grade (Sigma) was used
as the external standard.

2.2 Apparatus

The ultrasound-assisted extractions (UAE) were performed with an ultrasonic

device (USC 2800A, 40 kHz, Unique®) equipped with a digital timer and a temperature
controller. A Waters HPLC Alliance with e2695 separation module, 2998 photodiode array
detector (PDA), and software Empower (version 2.0) were used. Chromatographic
separations were performed using a Zorbax Eclipse Plus Agilent C18 column (250 mm X
4.6 mm, 5 µm) and a Phenomenex C18 guard cartridge system (30 mm X 4 mm, 4 µm).
The mobile phases were filtered through a PVDF membrane (0.45 µm, Merck®) and
degassed using an ultrasonic bath. Samples and analytical standard solutions were
previously filtered through a 0.45 µm PTFE membrane (Millex®).

2.3 Plant material

Dried whole fruits of A. indica were acquired from cultures performed at the Monte
Belo farm, located in the municipality of Alto do Rodrigues in the state of Rio Grande do
Norte, Brazil. The authenticity of the plant material was confirmed by a qualified
professional. Before acquisition, the fruit lot was kept in the farm warehouses and kept dry,
cool, and protected from light for approximately a year.
The fruits were separated from the soil by picking and sieving processes, washed in
water and dried at 40 °C in a circulating air oven for three days. They were then crushed in
a hammer mill (Ecirtec) and packed in containers protected from light and moisture. The
pharmacognostic characterization [28] of plant material revealed a moisture content of
4.73% (±0.066), total ash of 4.58% (±0.046), intumescence index of 3.26 mL, and particle
size of 1055 µm.

2.4 Ultrasound-assisted extraction process

2.4.1 Previous assays for UAE optimization

To test the main factors affecting the azadirachtin extraction efficiency from
botanical material and propose the conditions to be optimized using UAE, experiments
were conducted with a material-to-solvent ratio of 0.2 to 0.3 g mL-1, ethanol concentration
of 50 to 70% (w/w), temperature of 30 to 50°C, and extraction time fixed at 30 min. The
experiments were carried out following a Box–Behnken design associated with the
response surface methodology (RSM) with three factors and three levels. The conditions
that provided the highest concentrations of azadirachtin, quantified by HPLC-PDA (section
2.5), formed the basis for optimization design.

2.4.2 Experimental design

The optimization of the azadirachtin UAE process in dried fruits of A. indica was
performed from the experiments set out in the Box-Behnken design, as shown in Table 1.
Three factors that affect extraction efficiency in three levels (3 3) were investigated: ethanol
concentrations of 50, 70, and 90% (w/w, x1); extraction temperatures of 30, 45, and 60°C
(x2), and material-to-solvent ratios of 0.25, 0.4, and 0.55 g mL-1 (x3). The complete design
was carried out in random order and consisted of 15 combinations, including three
replicates at the central point (Table 2). A second-order polynomial regression model was
used to express the azadirachtin content as a function of the independent variables (Eq.
1).

∑ ∑ ∑∑ (Eq. 1)

where y is the predicted response, β0 is a constant, and βi, βii, and βij are the linear,
quadratic and interactive coefficients of the model, respectively. Accordingly, xi and xj
represent the levels of the independent variables.

[Insert Table 1]

RSM was used to investigate the influence of the three independent variables on
the azadirachtin content, check the predictive capability of the model, and establish the
best extraction conditions within the evaluated intervals. The experimental results were
analyzed using Statistic software version 7.0 [29], wherein two-way linear and quadratic
interactions have been included only for the factors with effects that were significant (p
<0.05, and in certain cases, p <0.1).

2.5 HPLC-PDA analysis of extracts

 Azadirachtin quantification in the extracts obtained from the optimization
experiments was performed using HPLC-PDA, and the choice of the analytical method is
described below. We carried out the linear regression of the external standard, and the
equation of the analytical curve was obtained from six concentration levels of the
azadirachtin standard (5000, 2000, 1000, 500, 250, and 125 µg ml-1) in three replicates.
The HPLC-PDA analytical method for azadirachtin quantification was performed
after testing some chromatographic systems most frequently cited in the literature for
identifying and quantifying this marker, as well as small variations shown in Table 2. To
evaluate the test-conditions, the azadirachtin standard (1000 μg ml-1 in methanol) and
ethanol (70% w/w) extract of the dried fruits of A. indica (EEF) obtained by UAE (material-
to-solvent ratio of 0.25 g mL-1, temperature of 30 °C, and extraction time of 30 minutes)
were injected. The injection volume was 10 µL, and the experiments were performed in
triplicate.

[Insert Table 2]

The number 1 chromatographic system was chosen, which comprised

acetonitrile/water (40:60) as the mobile phase, a detection wavelength of 214 nm and flow
rate of 1 mL min-1; the column oven temperature was maintained at 30 °C. This method
was the simplest and showed the azadirachtin peak of higher intensity and the system
suitability parameters for an azadirachtin peak (resolution - Rs, tailing factor - TF, and
number of theoretical plates - N) according to prerogatives of the United States
Pharmacopeia and Food and Drug Administration [30], expressed as mean values from six
determinations (± SD) (Table 3).

[Insert Table 3]

2.6 HPLC-PDA method validation

The method was validated according to the International Conference on the

Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals
for Human Use [31] and the Brazilian legislation [32].
The sample consisted of ethanol extract obtained by exhaustive percolation of 500
g of plant material in approximately 3.5 L of ethanol 70% (w/w) followed by concentration
in a Buchi® rotary evaporator (R-220SE model) under vacuum (40 °C) to obtain a
material-to-solvent ratio of 0.6 g mL-1. The concentrate liquid extract (CLE) showed an
ethanol content of 49% (w/w), relative density of 0.98 g mL-1 (20 °C), viscosity of 7.8 mPas
(Rheometro Brookfield® Model DV-III+, at 27 °C), a solid residue content of 17.7% (w/w),
and pH 5.4.

2.6.1 Selectivity
The selectivity of the method was evaluated comparing the chromatograms of a
blank (methanol), sample solution, mobile phase, and standard. The spectral similarity of
azadirachtin peaks in the standard and sample was also evaluated by comparing the UV
spectra in the wavelength range of 190 to 400 nm.

2.6.2 Linearity
The linearity was determined by the calibration curves obtained from HPLC analysis
at six concentration levels of the azadirachtin standard (1000, 500, 250, 125, 62.5, and
31.25 µg mL-1) in methanol. Each point was prepared in triplicate, and the calibration curve
was fitted by linear regression from the correlation between the peak areas and the
concentration of the standard. The linear regression coefficients (r) and analysis of
variance (ANOVA) were calculated.

2.6.3 Limit of detection and limit of quantification

The limit of detection (LOD) and limit of quantification (LOQ) were calculated based
on the standard deviation (SDb) of the intercept with the y-axis and the slope of the
calibration curve (S) according to Eqs. (2) and (3).

LOD=SDbX3 (Eq. 2)
S
LOQ=SDbX10 (Eq. 3)
S

2.6.4 Precision
The precision was evaluated at two levels: repeatability (intra-day) and intermediate
precision (inter-day), using the relative standard deviation (RSD) as criteria. The
repeatability of the method was verified from 9 injections of the test concentrations,
comprising high, mean and low concentrations of the standard linear range, namely, three
injections of each test concentration. The intermediate precision was evaluated by this
same process, performed on two different days by different analysts.

2.6.5 Accuracy
The accuracy was determined by recovery analysis. CLE methanol solutions were
prepared, in triplicate, at three concentration levels corresponding to 80, 100 and 120% of
the standard concentration in the linear range, with and without the addition of a known
amount of the azadirachtin standard (125 μg mL-1). The accuracy was calculated for each
level through the ratio between the average experimental concentration and theoretical
concentration of the added standard according to Equation (4).

Accuracy=sample conc. with standard – sample conc. without standard X 100% (Eq. 4)
standard theoretical concentration

2.6.6 Robustness
The robustness of the method was evaluated by analyzing the results of the CLE
azadirachtin content (147 mg mL-1, in methanol) obtained from the original conditions of
analysis and modified conditions. The injections were performed in triplicate and the
results were evaluated by the RSD calculation. The following parameters were changed:
column lot, column oven temperature of 30 °C to 29 °C and 31 °C, and the acetonitrile
manufacturer used in the mobile phase.

2.6.7 CLE azadirachtin quantification

The average calibration curve and the resulting equation of the standard linear
regression were used to quantify the CLE azadirachtin.

3. Results and Discussion

3.1 Previous assays for UAE optimization

Previous assays indicated the following influences of the evaluated parameters on

the azadirachtin content: significant quadratic effect (p = 0.034) of temperature in the
range of 30-50 °C, significant quadratic effect (p = 0.015) of ethanol concentration in the
range of 50% to 70% (w/w), and significant positive linear effect (p = 0.0002) of material-
to-solvent ratio in the range of 0.2 to 0.3 g mL-1. However, there were no significant
interactions among the parameters evaluated. The Response Surfaces (data not shown)
generated from these data did not allow the determination of extraction optimal conditions,
indicating that new experiments with different levels of factors should be performed, whose
results are presented below.

3.2 Effect of extraction parameters on azadirachtin content

Table 4 shows the azadirachtin contents of extracts from 15 experiments generated

by the Box-Behnken design. The multiple linear regression analysis using the quadratic
polynomial model (Eq. 1) was performed based on the results shown in Table 2. The
quadratic model contribution was significant (p = 0.000658), and R2 and R2 adj values of
0.97 and 0.89, respectively, confirm the model adequacy. Table 5 shows the results of
analysis of variance (ANOVA), as calculated based on the pure error. The lack-of-fit was
not significant (p>0.05), which indicates the suitability of the model to accurately predict
the variation.

[Insert Table 4]

[Insert Table 5]

The Table 5 data shows a significant primary linear effect (p<0.05) of the material-
to-solvent ratio (x3) and a significant primary quadratic effect (p<0.1) of the temperature on
the azadirachtin content. The second order interactions were significant (p<0.1) for ethanol
concentration (x1)/temperature (x2), ethanol concentration (x1)/material-to-solvent ratio (x3),
and temperature (x2)/ material-to-solvent ratio (x3).
Figure 2 shows the correlation between the data predicted by the model and the
observed data, showing high correlation between both.
The Box-Behnken design selects points from a three level factorial design, which
allows efficient estimation of the first and second order coefficients of the mathematical
model with the smallest number of experiments in comparison with other factorial models
[33- 35].
Figures 3 A-C present the response surface and contour plots for the influences of
UAE parameters on azadirachtin content. As shown in Figure 3A, the maximum extraction
of azadirachtin was obtained in ethanol concentrations (x1) of the range 70% to 90% (w/w)
and material-to-solvent ratio (x3) of 0.55 g mL-1. The azadirachtin is soluble in polar organic
solvents, such as ethanol and methanol, and slightly soluble in water [36], which may
explain the superior efficiency of its extraction by solutions with higher ethanol content.
Furthermore, the hydroethanolic solutions are widely used in extractive processes
precisely because of their extraction efficiency and low toxicity compared to other organic
solvents.
The temperature can impact the extractive processes, leading to an increase in the
substances extraction while causing losses of those that are thermosensitive. Once
thermogravimetric analyses indicated that the azadirachtin decomposition only occurs at
300°C [37] and its melting point is located between 154-158°C [36], we decided to
investigate the temperature influence on azadirachtin content in the UAE. Figure 3B shows
that the greatest azadirachtin contents were observed at temperatures near 30°C and a
material-to-solvent ratio of 0.55 g mL-1. In Figure 3C, the increased extraction of
azadirachtin is observed with the combination of temperatures up to 30°C and any of the
ethanol concentrations of the experiment. Moreover, evidencing the quadratic effect
identified by the model, higher temperatures (~60°C) increased azadirachtin extraction
when ethanol concentrations were below 75%.
The parameter material-to-solvent ratio (x3), analyzed in the response surface
graphs (Figure 3A-B), indicates that its maximum point is outside the experimental area. In
these cases, increased levels should be used in new experimental designs to obtain the
optimal value. However, this is not feasible experimentally due to the high intumescence of
the plant material.
According to Bezerra et al. [35], if the experimental region cannot be displaced by
physical or instrumental reasons, research must seek out the best conditions inside the
studied experimental conditions by visual inspection. Therefore, the best conditions for
UAE of azadirachtin from whole dried fruit of A. indica inside the investigated ranges and
acquired from the SRM general optimization function were: ethanol concentration range of
75 – 80% (w/w), temperature of 30°C, and material-to-solvent ratio of 0.55 g mL-1. Under
these conditions, the value predicted for azadirachtin content was 2607.3 μg mL-1. These
conditions were validated in independent experiments conducted in triplicate, giving an
average of azadirachtin content of 2375.6 µg mL-1. This corresponds to 91.1% of the
predicted value, demonstrating the validity of the model in predicting the phenomenon
studied.

3.3 HPLC-PDA method validation

 Figures 4 A-B show the chromatographic profile and UV spectrum (190-400 nm) of
azadirachtin obtained from the HPLC-PDA analysis of CLE and the azadirachtin reference
standard. The comparison of the CLE (Figure 4A), azadirachtin standard (Figure 4B), and
methanol (Figure 4C) chromatographic profiles, as well as the spectral similarity of
azadirachtin peaks at CLE (Figure 4A) and the standard (Figure 4B), show the selectivity
of the method. Interfering substances are not observed at the retention time of
azadirachtin, and the UV spectrum of azadirachtin in the sample is demonstrated to be
identical to the standard.
The results of the method linearity evaluation at six concentration levels are
presented in Table 6.

[Insert Table 6]

The calibration curve for azadirachtin was linear in the range of 31.25 – 1000
µg.mL-1 (Figure 5). The representative linear equation was: y = 912.53x + 9269.1 (N = 6; r
= 0.9998; RSD = 1.43%).
A correlation coefficient value close to unity is not enough to prove the linear
correlation and, hence, a test for lack-of-fit should be applied to evaluate the variance of
the residual values [38]. The ANOVA for the azadirachtin linearity is shown in Table 7. The
calculated F value for the lack-of-fit was smaller than the tabulated F value for a
confidence level of 95% (p = 0.05), demonstrating that the linear regression showed no
lack-of-fit.

[Insert Table 7]

The relative standard deviation (RSD%) for the slope of the azadirachtin calibration
curve was 1.43%. This value is within the limits set by ICH and ANVISA [31, 32], which
should not exceed 5%. The LOD value, understood as the lowest absolute concentration
of the analyte in the sample that can be detected but not necessarily quantified as an
exact value, in the indicated experimental conditions was 8.06 μg mL-1 (0.008 mg mL-1).
The LOQ value, understood as the lowest amount of analyte in a sample and which can be
quantitatively determined with suitable precision and accuracy under the indicated
experimental conditions, was 26.88 μg mL-1 (0.0268 mg mL-1).
The results of method precision, at repeatability and intermediate precision levels,
are shown in Table 8. For both tests, the RSD was less than 5% among triplicates of low,
medium and high concentrations, i.e., on the nine determinations, as recommended by
ANVISA [32]. The repeatability demonstrates the correlation between the results of
successive measurements of the same method performed under the same conditions in a
short time. Meanwhile, the intermediate precision aims to verify if, in the same laboratory,
the method provides the same results although run by different analysts on different days.
The results concerning the accuracy of the analytical method by the recovery test
are shown in Table 9. The recovery ranged from 96.96% to 104.66% with an average of
100.58% and RSD of 2.99%. The recovery test measures the amount of the substance of
interest present or added in the analytical portion of the test material that is extracted and
capable of being measured [39]. According to Ribani et al. [40], the acceptable recovery
intervals depend on the analytical complexity and sample, and they admit that the amounts
may range from 50 to 120% with an accuracy of ± 15%.

[Insert Table 8]

[Insert Table 9]

Variations in the column oven temperature, manufacturer of acetonitrile used in the

mobile phase composition, and column lot resulted in RSD values below 5% for retention
time, peak area, and azadirachtin content (Table 10), demonstrating the method’s
robustness. The robustness of an analytical method assesses its ability to resist small and
deliberate variations of the analytical parameters, indicating your confidence during normal
use [32]. The demonstration of robustness is critical in the transference of the analytical
process to other laboratories [41].

[Insert Table 10]

3.4 Comparison between azadirachtin contents in percolated extract and extract from UAE

The percentage of azadirachtin in the extract obtained from UAE, under optimal
conditions set out in the RSM [ethanol concentration range of 75 - 80% (w/w) at 30°C and
material-to-solvent ratio of 0.55 g mL-1], was 0.431% (w/w). On the other hand, the
average percentage of azadirachtin in the percolated extract was 0.12% (w/w), as noted in
the validation steps of the analytical method. These results confirm the superior efficiency
of the UAE, demonstrating that it can be safely used as a fast, inexpensive and simple
extractive method for the preparation of samples for HPLC analysis from fruits of A. indica.
Yolmeh et al. [26] reached similar findings in the UAE of natural pigments from annatto
seeds (Bixa orellana L.).

4. Conclusion

The results revealed that the UAE is an effective technique for extracting the
azadirachtin from A. indica fruit compared to percolation, which is a conventional
extraction method that requires more time and higher consumption of the solvent. The
RSM has been successfully employed in determining optimum conditions for ultrasound-
assisted extraction of azadirachtin, and the following conditions were established: ethanol
concentration of 80% (w/w) at 30°C and a material-to-solvent ratio of 0.55 g ml-1.
The HPLC-PDA analytical method used for azadirachtin quantification was validated
according to the ICH guidelines and Brazilian regulations and demonstrated to be simple,
selective, linear, precise, accurate and robust, being useful for the analysis of this marker
in A. indica fruit extracts.

Acknowledgments

The authors are grateful to Conselho Nacional de Ciência e Tecnologia (CNPq),

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação
de Amparo à Pesquisa do Estado de Goiás (FAPEG) for their financial support.

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Figures

Figure 1 – The azadirachtin chemical structure.

Figure 2 – Correlation between experimental values (observed values) and predicted values (calculated
using statistical model) for the azadirachtin content.

A B C

Figure 3 – Response surface for azadirachtin content from UAE experiments of A. indica fruits.
 0,40 9,650 azadiractina
213,4

Azadirachtin
A
0,010

RT= 9,65

AU
0,005
AU

0,20
363,3382,4
0,000
200,00 250,00 300,00 350,00 400,00
nm

0,00

2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00
Minutes

0,06 9,547 Extracted

213,4
Azadirachtin
B
0,030

0,04
RT= 9,54 0,020

AU
AU

0,010

0,02 0,000
392,0

200,00 250,00 300,00 350,00 400,00

nm

0,00
0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00
Minutes

1,00
C
AU

0,50

0,00
0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00
Minutes

Figure 4 – HPLC-PDA chromatographic profiles of: CLE (A); azadirachtin reference standard (B); and
methanol (C), followed by UV spectra of azadirachtin (190-400 nm).
Chromatographic conditions: C18 column (250 x 4.6 mm, 5 µm); mobile phase acetonitrile/water (60:40);
-1
flow rate of 1.0 ml min ; column temperature of 30 °C; λ = 214 nm; injection volume of 10 µL.

1000000
900000
800000
Peak area (µAU.s)

700000
600000
500000
400000
300000 y = 912,53x + 9269,1
200000 R² = 0,9997
R = 0,9998
100000
0
0 200 400 600 800 1000 1200
Azadirachtin content (µg mL-1)

-1
Figure 5 - Azadirachtin calibration curve in the concentration range of 31.25-1000 µg mL .
Tables

Table 1 – Levels of variables for the experimental design.

Levels

Symbols Independent variables -1 0 +1

x1 Ethanol (%, w/w) 50 70 90

x2 Temperature (°C) 30 45 60

x3 Material-to-solvent ratio (g mL-1) 0.25 0.4 0.55

Table 2 – HPLC-PDA chromatographic systems tested in the azadirachtin separation from

fruit ethanol extract of A. indica.

System Mobile phase Flow (mL min-1) Wavelength (nm) Reference

1 Acetonitrile/water (40:60) 1 214 and 217 [10, 16]

2 Acetonitrile/water (35:65) 1 217 [19]

3 Methanol/water (90:10) 1 214 [13]
4 Acetonitrile/methanol/water 1 210 -
(30:30:40)
5 Methanol/water (50:50) 1 214 -

Table 3 - Mean (± SD) of the system suitability parameters for chromatographic system 1,
obtained from six azadirachtin determinations in the EEF of A. indica.

System suitability
Tailing factor (TF) Resolution (Rs) Theoretical plates (N)

Sample azadirachtin
1.16 (±0,13) 2.55 (±0,21) 15020.02 (±618,20)
peak

Literature
recommendations ≤ 2,000 ≥ 2,000 > 2.000,000
[30]
Table 4 – Box-Behnken design arrangement for UAE parameters (independent variables,
x1, x2, and x3) and the response of the azadirachtin content (dependent variable).

Run number x1 (%) x2 (°C) x3 (g mL-1) Azadirachtin (µg mL-1)

1 50 30 0.40 1325.585
2 90 30 0.40 1344.883
3 50 60 0.40 1503.609
4 90 60 0.40 1190.742
5 50 45 0.25 630.184
6 90 45 0.25 615.975
7 50 45 0.55 1277.748
8 90 45 0.55 2102.092
9 70 30 0.25 684.386
10 70 60 0.25 694.430
11 70 30 0.55 2691.829
12 70 60 0.55 1908.519
13 70 45 0.40 1414.271
14 70 45 0.40 1353.587
15 70 45 0.40 1185.835

x1= ethanol; x2= temperature; x3= material-to-solvent ratio

Table 5 – Analysis of variance (ANOVA) for the quadratic polynomial regression model.

Sum of Degrees of Mean

Factor F value p
squares freedom squares
x1 2486 1 2486 0.1776 0.714428
x2 70197 1 70197 5.0137 0.154516
x3 3678028 1 3678028 262.6966 0.003785*
x12 91583 1 91583 6.5412 0.124877
x22 120696 1 120696 8.6205 0.099065**
x32 56 1 56 0.0040 0.955197
x1x2 27583 1 27583 1.9701 0.295561
x1x22 152270 1 152270 10.8756 0.080942**
x1x3 175793 1 175793 12.5557 0.071239**
x2x3 157352 1 157352 11.2386 0.078628**
x22x3 147928 1 147928 10.5655 0.083030**
Lack-of-fit 79431 1 79431 5.6732 0.140144
Pure error 28002 2 14001
Sum of squares
4686629 14
total

x1= ethanol; x2= temperature; x3= material-to-solvent ratio

*p<0.05; **p<0.1

Table 6 – Summary of the calibration curve parameters of azadirachtin.

Azadirachtin
Linear range (µg mL-1) 1000 – 31.25
-1
Limit of detection (µg mL ) 8.06
Limit of quantification (µg mL-1) 26.88
Linear regression data*
N 6
Slope (a) 912.53
Standard deviation of slope 13.09
Relative standard deviation of slope (%) 1.43
y-axis intercept (b) 9269.1
Linear correlation coefficient (r) 0.9998
* y = ax + b, where x is the compound concentration and y is the peak area.

Table 7 – ANOVA data for azadirachtin linearity.

Azadirachtin
DF SS MS F Ftab
Model 1 1.7163 x 1012 1.71626 x 1012 43336.141 4.494
Residual 16 914163333 57135208.29
Lack-of-fit 4 438921071 109730267.7 2.770 3.259
Pure error 12 475242262 39603521.83
DF = Degrees of freedom; SS = Sum of Squares; MS = Mean Squares; F = calculated F value; Ftab =
tabulated F value.

Table 8 - Data of HPLC analytical method precision at repeatability and intermediate

precision levels for the azadirachtin quantification in the CLE.

Concentration level of CLE Azadirachtin

Area Azadirachtin
the linear range of the concentration content
-1 (µAU.s) (%)
method (mg mL ) (mg mL-1)
Intra-day (day 1, analyst 1)
Low 39.2 49803 0.044 0.11
39.2 49368 0.044 0.11
39.2 49029 0.044 0.11
Medium 147 176350 0.183 0.12
147 173755 0.180 0.12
147 174544 0.181 0.12
High 539 589156 0.635 0.12
539 592684 0.639 0.12
539 617808 0.667 0.12

RSD% 4.47

Intra-day (day 2, analyst 2)

Low 39.2 49354 0.044 0.11
39.2 49986 0.045 0.11
39.2 49855 0.044 0.11
Medium 147 172167 0.179 0.12
147 171621 0.178 0.12
147 170464 0.177 0.12
High 539 630326 0.681 0.13
539 633677 0.684 0.13
539 630563 0.681 0.13

RSD% 4.87

Inter-day (Intermediate precision)

RSD% 4.59

RSD% = Relative Standard Deviation

Table 9 – Data of HPLC analytical method accuracy for the azadirachtin quantification in
the CLE.
Concentration Azadirachtin area Azadirachtin content
CLE Azadirachtin -1
level of the (µAU.s) in the ELC (mg mL ) in the ELC Recovery
concentration area (µAU.s)
linear range of -1 + azadirachtin + azadirachtin (%)
(mg mL ) in the CLE
the method standard standard
Low 39.2 22097 141484 0.1448 104.6646
39.2 22983 141716 0.1451 104.0912
39.2 23403 142012 0.1454 103.9825
Medium 147 98715 209318 0.2192 96.9638
147 97239 208320 0.2181 97.3828
147 93028 206315 0.2159 99.3168
High 539 319414 434432 0.4659 100.8343
539 319981 434094 0.4655 100.0409
539 320427 432239 0.4635 98.0237
-1
Theoretical concentration of azadirachtin standard (mg mL ) 0.125

100.58
Average recovery (RSD%)
(2.99)

DPR% = Desvio Padrão Relativo

Table 10 – Chromatographic parameters on the robustness testing of the HPLC analytical

method for azadirachtin quantification in the CLE.

Parameter Average (RSD% intra)

Rt Area Azadirachtin (%,
m/m)
Column oven temperature (°C)
29 9.695 (0.29) 176771.3 (0.435) 0.124 (0.459)
30 9.623 (0.19) 174084.0 (0.821) 0.122 (0.867)
31 9.710 (0.136) 174769.7 (0.986) 0.123 (1.041)
RSD% inter 0.457 0.965 1.019

Acetonitrile manufacturer
A 9.623 (0.198) 174084 (0.821) 0.122 (0.867)
B 9.522 (0.281) 176350 (0.069) 0.124 (0.073)
RSD% inter 0.617 0.877 0.926

Column Lot
B11165 9.623 (0,198) 174084 (0.821) 0.122 (0.867)
B14007 8.960 (0,099) 166696.3 (0.501) 0.117 (0.530)
RSD% inter 3.91 2.453 2.594

Rt = Retention time; RSD% = Relative Standard Deviation

• Azadirachta
indica dried
fruits

• size of
pulverized
particles: 1055
µm

• Ethanol
concentration of 50,
70, and 90% (w/w)
• temperatures of 30,
45, and 60°C
• material-to-solvent
ratios of 0.25, 0.4,
and 0.55 g.mL-1

• Azadirachtin
content

• HPLC-PDA
analytical
method
validation