RESEARCH COMMUNICATIONS

Characterization and testing of fine ester group in Aza isomerizes into Z-2-methylbut-2-
enoate ester when irradiated as a thin film under UV light
powder formulation of whole neem at 254 nm, due to the presence of -electrons. Kumar et
fruits al.6 studied the stability of Aza in different solid carriers
and found that degradation is least in attapulgite.
Sonali Tajane1 , Praful Dadhe1 , Sayaji Mehetre2 In this study, whole neem fruits are used in their origi-
nal form without extracting Aza separately. This method
and Sachin Mandavgane1,*
1
ensures better Aza stability as well as effective utilization
Department of Chemical Engineering,
of other limonoids in neem fruits. According to Verkerk
Visvesvaraya National Institute of Technology, Nagpur 440 010, India
2
Nuclear Agriculture and Biotechnology Division, and Write7, presence of other limonoids in neem seeds
Bhabha Atomic Research Centre, Mumbai 400 085, India increases the biological activity of pure azadirachtin.
Whole neem fruits are dried and ground with dolomite
Azadirachtin (Aza) is a key ingredient of neem-based into fine powder using a special technique developed by
pesticides. However, use of neem pesticides is limited them. This method reduces particle size and creates new
due to storage instability of Aza. In this work, free- surface area, which in turn increases the availability of
flowing fine powder of whole dry neem fruits (powder Aza on the surface of the particles. Dolomite acts as an
neem formulation, PNF) is developed without sepa- inert material and does not have any effect on plant
rately extracting Aza. The optimal particle size was growth except increasing the leaf size8,9. Normally, oil
found to be −44 + 60 mesh. PNF is characterized by
seeds cannot be converted into fine powder because they
Fourier transform infrared, X-ray diffraction, Brun-
auer–Emmett–Teller surface area, particle-size distri- contain oil, and therefore the neem fruit powder is limited
bution and scanning electron microscope. Stability of to coarser size, which results in a comparatively small
Aza was found to be improved and it was assessed by surface area. This problem is overcome by grinding dry
studying the effect of particle size, temperature, UV neem fruits with dolomite, which absorb or adsorb re-
light exposure and release study in buffered and natu- leased oil and make PNF free flowing. PNF produces a
ral water samples. sustained release effect since the ingredients are gradually
released, thus ensuring long-term protection from insects.
Keywords: Azadirachtin, characterization, free-flowing PNF could also be directly dusted near the root of crops
powder, particle-size, stability. or may be sprayed directly mixed with water, on the
leaves. Thus, the insecticidal effect could be much
BIOPESTICIDES are obtained from plants and microorgan- stronger and long lasting.
isms and have gained significant attention in recent times Dry neem fruit and dolomite powder were purchased
due to their harmful effects of chemicals. Besides, these from a local market. Aza (95% purity) was purchased
compounds are more selective and biodegradable. The from Sigma Chemical Co. Methanol, water and acetoni-
most commonly used biopesticides are neem-based. Since trile (all HPLC grade) were purchased from Fisher Scien-
ancient times, different parts of Azadirachta indica tific India.
(neem) tree such as leaves, bark, flowers, fruits and seeds Pre-treatment of whole dry neem fruit included wash-
are used to control pests and for various other medicinal ing, cleaning and drying. Cleaned dry fruits were then
applications. Azadirachtin (Aza) is a key ingredient of pulverized with dolomite powder (85 mesh size) with
neem-based biopesticide responsible for inhibition of 1 : 1 ratio by weight in a hammer mill (RPM-9000; HP-2
pests. It is a powerful insect antifeedant and growth- Shubh Micro Baby Pulverizer, India). After pulverization,
regulating substance; besides controlling pests, neem PNF was sieved and its moisture content was determined
extracts are also found to inhibit nitrification and retard according to the Bureau of Indian Standards (IS 3579-
nematode growth1,2. However, use of neem pesticides is 1966). Moisture contents in all samples were 6%  0.5%
limited owing to Aza storage instability. (wt/wt). Figure 1 shows dried whole neem fruit, dolomite
The three major factors that influence instability of powder and PNF.
Aza are degradation in water, pH sensitivity and photo- Each study was conducted by analysing the concentra-
degradation. Szeto et al. 3 studied hydrolysis of Aza in tion of Aza in PNF, which was determined by HPLC
natural water and aqueous buffered solution, and found (Water Make Model no. 515) method10–12. The chroma-
that these active materials are non-persistent in aquatic tographic determination of Aza from different PNF sam-
environment. Andrew et al.4 suggested that Aza is highly ples was achieved using a C-18 analytical column with a
stable in mildly acidic (pH 4 and 6) aqueous solutions at water–acetonitrile mixture (65 : 35) as the mobile phase
room temperature, but it is unstable in mildly alkaline (flow rate 1 ml/min; UV detector at 214 nm). A 10 l
and strongly acidic solutions. Durege et al.5 noted that sample was then directly injected into the column. The
Aza is highly photolabile. The (E)-2-methylbut-2-enoate retention time of Aza was 18.49 min (ref. 12). A stock
solution (40 ppm) of standard Aza (95% purity) was pre-
*For correspondence. (e-mail: [email protected]) pared in methanol and stored in a volumetric flask.
1942 CURRENT SCIENCE, VOL. 112, NO. 9, 10 MAY 2017
 RESEARCH COMMUNICATIONS

Figure 1. Free-flowing fine powder of whole dry neem fruits.

taining Aza was separated. The extract was then concen-

trated to 10 ml by rotary evaporation (Jain Scientific
Glass Work). The concentrated sample was passed
through a fluorescein column to remove colour impurities
and then passed through a syringe filter with a pore
diameter of 0.456 m (Millipore, USA), diluted, and
finally loaded onto the HPLC column 13. Before conduct-
ing the experiment, the initial concentration of Aza in
PNF was determined each time, in mg per 10 g of PNF
sample. HPLC peaks of standard Aza (95% pure) in me-
thanol and the extract of PNF in methanol are shown in
Figure 2 a and b respectively.
PNF was characterized by different techniques. Particle-
size distribution was analysed using Fritsch particle sizer
(ANALYSETTE 22) to obtain size fractions of PNF.
FTIR of dry neem fruit, dolomite and PNF were recorded
using a Perkin Elmer spectrometer. The spectral data
were used to compare the functional groups of raw mate-
rials and PNF. The XRD patterns were recorded on a
Philips X’Pert Pro PANalytical PW3040/60 diffractometer
to obtain information about crystallographic structures.
Figure 2. HPLC peak of Aza with retention time of 18.496; a, Stan-
dard Aza sample in methanol; b, Crude extract of PNF sample in SEM analysis (JEOL 6380A/JFC 1600) was carried out to
methanol. study the surface morphology of PNF. BET surface area
of dry neem fruit, dolomite and PNF was determined
using a surface area analyser (Smart Sorb 92/93).
The flask was then kept inside a refrigerator (below PNF contains Aza in its natural form as well as other
0C). Using this stock solution, Aza solutions of different limonoids. Effect of various parameters, such as particle-
concentrations (5, 10, 15 and 25 ppm) were prepared. size, temperature and UV exposure, on the stability of
These solutions were analysed by HPLC. Retention time PNF was studied. In addition, we also studied the release
of pure Aza (i.e. 18.49 min) and the peak area of different of PNF in buffered and natural water.
solutions were determined. A calibration plot was used to Five fractions of different sizes of PNF were taken for
correlate the concentration of unknown sample. Aza con- this study (Table 1). Each fraction was quantified by Aza
tent in PNF was determined by ultrasonic assisted extrac- available on the particle surface and Aza present inside
tion of the sample in methanol using a water bath the PNF particles. Aza content on the surface was deter-
sonication process (Ultrasonics Pvt Ltd, Mumbai, India). mined by sonication-assisted extraction at ambient tem-
About 10 g of the PNF sample was mixed with 100 ml of perature for 30 min. Aza present inside the particles was
methanol (HPLC grade) and sonicated for 30 min. This quantified by Soxhlet extraction in methanol. In brief,
sample was then centrifuged and the extract phase con- this involved the following steps: 10 g of PNF wrapped in
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Table 1. Surface and total extractable Aza in PNF samples with different particle sizes

Sieve mesh no. Mean size Surface azadirachtin Total extractable azadirachtin % Aza available on
(as per BSS) (m) (mg/10 g of sample)* (a) (mg/10 g of sample)* (b) surface (a/b)  100

−16 + 25 957 10.7 22.3 48

−25 + 44 533 13.6 24.3 56
−44 + 60 303 20.9 26.9 77.7
−60 + 85 215 21.8 27.1 80.5
−85 + 100 165 23.1 28.6 80.8

*Mean of three replicates determined in five samples. B.S.S – British Stander Sieves BSS 410-2000.

Table 2. Release study in different aqueous solutions

% Aza remaining % Aza release

Concentration (ppm) in PNF cake in filtrate

Buffered solution of pH 9.2 NF NF

Buffered solution of pH 4.0 100 NF
NMC water sample pH 8.02 82.9 17.02
Well water sample pH 7.57 87.8 12.1

*The study was conducted for total period of 48 h in a dark compart-

ment with an average temperature of 23C. NMC, Nagpur Municipal
Corporation; NF, Not found (no peak appears at retention time of
18.46 min).

The release of PNF was studied using the following

four aqueous samples: buffer solutions of pH 9.2 and 4.2,
laboratory tap water (pH 7.57), and local municipal cor-
poration water supply (pH 8.02) (Table 2). About 10 g of
Figure 3. FTIR of dry neem fruit, dolomite and PNF.
PNF was mixed with 50 ml of each aqueous solution.
After 48 h, the solution was filtered, and the cake and
filtrate were collected separately. The cake was dried and
a filter paper was placed in a Soxhlet apparatus. About Aza content determined. The filtrate was completely
150 ml of methanol (HPLC grade) was added to carry out evaporated at 60C and the Aza content of the residue
the extraction at 64.7C for 4 h (ref. 12). The solvent was was determined.
recovered and the PNF extract in methanol was concen- It was observed that as the particle-size of PNF
trated to 10 ml. The concentrated sample was passed decreases, the percentage of Aza available on particle
through a fluorescein bed and then through a syringe fil- surface increases (Table 1). This is because, as the
ter. The clear sample was diluted and loaded onto the particle-size decreases, a new surface of PNF is created,
HPLC column. which increases the availability of Aza on the surface.
An optimal size fraction of PNF was taken to study the Approximately 48% of Aza is available for −16 + 25-
effect of temperature on its stability. Experiments were mesh size fraction of PNF and 80.8% is available for
conducted at 50C and 100C respectively. Initially, −85 + 100 fractions. In other words, finer the PNF parti-
100 g of PNF sample was taken in a petri dish and kept in cle, larger will be the Aza molecules available on the sur-
an oven at 50C. The sample was withdrawn at regular face. Table 1 shows that a size fraction of −44 + 60 mesh
intervals of 1, 3, 7 and 14 days. The concentration of Aza gives 77.7% surface Aza. By further decreasing the size
in each sample was determined by HPLC. The same to −85 + 100 mesh, Aza on the surface increased by only
experimental procedure was followed to study the effect 3.1%. Size reduction beyond 100 mesh is energy-
of temperature at 100C. intensive and moreover it is observed that below 100
To study the effect of UV radiation on PNF, 100 g of mesh size, there is no substantial increase in surface Aza.
powder was taken in a petri dish and kept in an UV When energy requirement for size reduction below 100
chamber (Biotechnic India-32, model no. 19222) for 14 mesh is compared with available Aza, it is found ineffi-
days. The UV lamp was placed at a distance of around cient. Similar observations were recorded by Jadeja et
18 cm from the PNF sample and a uniform temperature of al.12. For better surface Aza and proper energy for size
50C was maintained inside the chamber. The sample reduction, PNF powder of mesh size −44 + 60 was con-
was withdrawn at regular intervals of 1, 3, 7 and 14 days. sidered optimal.
Aza concentration in each sample was determined by FTIR spectra of pure neem fruit, dolomite and PNF are
HPLC. shown in Figure 3. FTIR spectra of neem fruit showed the
1944 CURRENT SCIENCE, VOL. 112, NO. 9, 10 MAY 2017
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Figure 4. XRD of (a) dry neem fruit, (b) dolomite and (c) PNF.

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Figure 5. SEM (i) 500 and (ii) 2000: a, dry neem fruit seed; b, dolomite; c, PNF.

following bonds and corresponding functional groups: study on neem leaves powder by Baljit Singh and Shar-
3326-hydroxyl O–H stretch (hydrogen bonded) in alco- ma14 . In the FTIR spectrum of pure dolomite, several
hol/phenol, 3306-C=H stretch in alkene, 2924-2854-C–H bands characteristic of carbonate group were observed.
stretch in alkene, 1746-C=O stretch in ester/saturated ali- The bands at 1464 and 1191 cm–1 corresponded to stret-
phatic, 1702-1672-C=O stretch in – unsaturated alde- ching vibrations of C–O; those at 881 and 729 cm–1 were
hyde and keton, 1465-C–H stretch in the aromatic ring, attributed to the out-of-plane and in-plane bending mode
1238-C–H stretch in alcohol carboxylic acid ester ether, of (CO3 )2 ion; and combinations of internal mode fre-
1161–1128, 1097-C=O stretch in alcohol carboxylic acid quencies with a lattice mode were observed at 2538 and
ester ether, 840-C–Cl stretch in alkyl halides, and 720-C– 2627 (1/cm) respectively15. In the FTIR spectrum of PNF,
H rock in alkanes. Similar bonds were also observed in a all the aforementioned peaks (neem and dolomite) were
1946 CURRENT SCIENCE, VOL. 112, NO. 9, 10 MAY 2017
 RESEARCH COMMUNICATIONS

Figure 6. Effect of temperature (50C and 100C) and UV radiation on PNF.

observed (Figure 3). This proved that there was no when PNF was treated at 50C, compared to that at
chemical reaction or thermal degradation of chemicals in 100C. These results confirmed an earlier study by
neem due to size reduction. Neem was present in PNF in Andrew et al.4. In general, if the on-field temperature
its very natural form. does not exceed 50C, then the PNF shows good stability.
XRD spectra of neem fruit (Figure 4 a) showed a However, PNF should be applied on crops during morn-
hump, which indicated the amorphous nature of neem ing hours. At 100C, it was observed that degradation of
powder. The d-spacing value of dolomite XRD (Figure Aza was slow in the initial period (up to 3 days) after
4 b) was compared with the reported d-spacing value16 which, it increased suddenly. The half-life period of Aza
and these were found to match. A sharp peak confirmed at 100C was 5 days.
the crystalline structure. XRD of PNF showed both crys- Effect of UV radiation on PNF showed that it was quite
talline and amorphous nature due to the presence of neem stable and its half-life period was more than 14 days.
powder and dolomite. Standard Aza has a reported half-life of 3.9 days when
SEM images provide insight into surface morphology. exposed to UV light11. Thus, it can be concluded that Aza
SEM images of pure dry neem fruit, dolomite and PNF in its natural form in PNF is more stable under UV radia-
are shown in Figure 5. The micrographs (500  magnifi- tion than the pure isolated Aza.
cation) showed the assembly of fine particles that were Release study of Aza in PNF in buffer solutions (pH
not in regular shape and size. At 2000  magnification, 9.2) showed that Aza was quite unstable and underwent
the SEM image of pure neem fruit powder appeared complete degradation in 48 h. Thus, its half-life is less
bulky/cloudy. This is because when the dry neem fruits than 48 h. In a pH 4.0 buffer solution, the amount of Aza
are crushed, oil is released and lumps are formed, which in the filtrate was negligible. This confirms that Aza was
causes cake formation. A rigid crystalline structure of completely present in cake and has a half-life of more
dolomite was observed at 2000  magnification. The than 48 h. In Municipal Corporation water pH 8.02 sample,
image of PNF at 2000  magnification indicated that the around 17% of Aza was present, whereas in laboratory
surface was free from oil. This is because the oil that tap water (pH 7.58), 12% of Aza was released from PNF.
oozes out during crushing is immediately adsorbed/ Thus, it can be concluded that the stability of Aza decreased
absorbed by dolomite and a homogeneous free-flowing with pH. These results demonstrate that at pH 7.58, re-
powder is obtained. lease of Aza was slow compared with that at pH 8.02, and
BET surface area of PNF was found to be 4.5 m2 /g. these also agreed with the study by Andrew et al.4.
Normally oil seeds cannot be converted into free-flowing The present study has been directed towards the use of
fine powder because of oil content. Neem fruit powder was whole dry neem fruits in their natural form and making it
limited to coarser size, which produced a comparatively more effective in terms of Aza content. Separation or iso-
small surface area. This problem was overcome by grinding lation of Aza can decrease their stability. This will also
dry neem fruit with dolomite, which removed oil and made add the need for unit operations such as depulping, and
the product free flowing with improved surface area. extraction with solvent, which unnecessarily increase the
Effect of temperature (50C and 100C) on PNF was manufacturing cost. Developing a simple technique for
studied (Figure 6). A minor reduction of Aza was noticed manufacturing PNF could improve its effectiveness as

CURRENT SCIENCE, VOL. 112, NO. 9, 10 MAY 2017 1947

RESEARCH COMMUNICATIONS
well as reduce the overall cost of manufacturing. Our Distribution and conservation status of
results show that PNF is economical, stable, and can have
a long-lasting effect on crops. the western tragopan
Tragopan melanocephalus in
1. Mohanty, S., Patra, A. K. and Chhonkar, P. K., Neem Jammu and Kashmir, India
(Azadirachta indica) seed kernel powder retards urease and nitrifi-
cation activities in different soils at contrasting moisture and tem- Riyaz Ahmad1,2 , Narayan Sharma2,3,
perature regimes. Bioresour. Technol., 2008, 99, 894–899.
2. Akhtar, M., Plant growth and nematode dynamics in response to
Upender Pacchnanda4 , Intesar Suhail4 ,
soil amendments with neem products, urea and compost. Biore- Kasturi Deb1, Yash Veer Bhatnagar2 and
sour. Technol., 1999, 69, 181–183. Rahul Kaul1
3. Szeto, S. Y. and Wan, M. T., Hydrolysis of Azadirachtin in buff- 1
Wildlife Trust of India, F-13, Sector 8, Noida 201 301, India
ered and natural water. J. Agric. Food Chem., 1996, 44, 1160– 2
Nature Conservation Foundation, 3076/5, IV Cross, Gokulam Park,
1163.
Mysuru 570 002, India
4. Andrew, P., Jarvis, S., Johnson, E. and David, M., Stability of the 3
Department of Environmental Biology and Wildlife Sciences,
natural insecticide Azadirachtin in aqueous and organic solvents.
Protection, Cotton College State University, Guwahati, 781 001, India
Pestic. Sci., 1998, 53, 217–222. 4
Department of Wildlife Protection, Rajbagh, Srinagar 190 001, India
5. Durege, P. and Johnson, S., Photodegradation of azadirachtin-A:
neem-based pesticide. Curr. Sci., 2000, 79, 1700–1703.
6. Kumar, J. and Parmar, B. S., Stabilization of Azadirachtin A in
In India, western tragopan is reported from Jammu
neem formulations: effect of some solid carriers, neem oil and sta- and Kashmir (J&K), Himachal Pradesh and Uttara-
bilizers. J. Agric. Food Chem., 1999, 47, 1735–1739. khand. We documented the current status and distri-
7. Verkerk, R. H. J. and Write, D. J., Biological activity of neem bution of western tragopan in J&K. We also predicted
seed kernel extract and synthetic azadirachtin against larvae of its potential distribution in the state. We used litera-
Plutella xylostellia L. Pestic. Sci., 1993, 37, 83–91. ture, field surveys and semi-structured questionnaire
8. Benjawan Chutichudet, P., Chutichudet, S. and Kasewsit, Effect of surveys to ascertain the distribution and conservation
dolomite application on plant growth international. J. Agric. Res., status of the pheasant species in J&K. Between 2007
2010, 5(9), 690–707. and 2011, we conducted counts of western tragopan in
9. Chen, G. C., He, Z. L., Stoffella, P. J., Yang, X. E., Yu, S. and
five areas: Tattakuti Wildlife Sanctuary, Khara Galli
Calvert, D., Use of dolomite phosphate rock (DPR) fertilizers to
reduce phosphorus leaching from sandy soil. Environ. Pollut.,
Conservation Reserve (CR), Limber Wildlife Sanctu-
2006, 139, 176–182. ary (WLS), Lacchipora WLS and Kazinag National
10. Kanth, M. S. and Sudaram, Azadirachtin biopesticide: a review of Park (NP) to assess its current status. We estimated
studies conducted on its analytical chemistry, environmental 113 callers of western tragopan from Kazinag NP,
behaviour and biological effects. J. Environ. Sci. Heal. B, 1996, Limber WLS, Lacchipora WLS, Tattakuti WLS and
31, 913–930. Khara Galli CR. We also discovered four new sites –
11. Dai, J., Yaylayan, V., Vijaya, A., Raghavan, G. S., Jocelyn, R. and Tattakuti WLS and Khara Galli CR (through direct
Pare, Extraction and colorimetric determination of azadirachtin- surveys), Noorpur Galli and Narian-Ratannard
related limonoid in neem seed. J. Agric. Food Chem., 1999, 47, (through secondary surveys) – of this species. We have
3738–3742.
confirmed the presence of western tragopan in Lac-
12. Jadeja, G. C., Maheshwari, R. C. and Naik, S. N., Extraction of
natural insecticide Azadirachtin from seed kernels using pressur-
chipora WLS and re-confirmed its presence in Padder,
ized hot solvent. J. Supercrit. Fluid, 2011, 56, 253–258. Bhadarwa and Sudh Mahadev. Our habitat model
13. Babic, S., Petrovic, M., Kastelan, M and Macan, Ultrasonic predicted potential distribution of western tragopan,
extraction of pesticides from soil. J. Chrom. A, 1998, 823, 3–9. adding few additional potential sites. There is an
14. Baljit Singh and Sharma, D. K., Development of a new controlled urgent need to plan long-term monitoring and initiate
pesticide delivery system based on neem leaf powder. J. Hazard. appropriate measures to conserve the species.
Mater., 2010, 177, 290–299.
15. Gunasekaran, S. and Anbalagan, G., Thermal analysis of natural Keywords: Conservation status, distribution, hunting,
dolomite. Bull. Mater. Sci., 2007, 30, 339–344. Tragopan melanocephalus.
16. Samtani, M., Skrzypczak-Janktun, E., Dollimore, D. and Alexan-
der, K., Thermal analysis of ground dolomite confirmation of
results using an X-ray powder diffraction methodology. Thermo- WESTERN tragopan Tragopan melanocephalus, a threat-
chim. Acta, 2001, 367–368, 297–309. ened pheasant species1, is endemic to the Western Hima-
laya 2. Its distribution ranges from northwestern Pakistan 3
through Kashmir4 into Himachal Pradesh (HP)5 and possi-
ACKNOWLEDGEMENTS. The authors thank the Board of Research
in Nuclear Sciences (BRNS), Department of Atomic Energy, GoI, for
bly the western parts of Uttarakhand6. The current global
funding. We also thank Dr B. D. Kulkarni and Subhash Pandit for their population of western tragopan is reported to be between
support. 2500 and 3500 individuals7, although earlier studies esti-
mated it to be around 5000 individuals2. The major
reasons for its global decline are habitat degradation and
Received 19 February 2016; revised accepted 11 January 2017

doi: 10.18520/cs/v112/i09/1942-1948 *For correspondence. (e-mail: [email protected])

1948 CURRENT SCIENCE, VOL. 112, NO. 9, 10 MAY 2017