Development of effervescent tablet formulation for rapid control of mosquito problem in early stages from different breeding sites

2.1 Neem oil- active principle

Neem oil was supplied by Theni, Gujarat, India. The percentage of constituents like triterpenoid compounds like Azadirachtin A + B (0.03%) and Nimbin (2.6%) along with fatty acids, specifically Omega-6 (5–15%), Omega-9 (22–25%), Stearic acid (9–24%) and Palmitic acid (12–15%). Specific gravity, saponification value and impurities & moisture content of neem oil are 0.921 g.ml^-1at 280.15 K, 203 mgKOHg⁻¹, and 0.17 g.g⁻¹ w/w respectively.

2.2 Disintegrating agent

D + Glucose purchased from sigma Aldrich of purity >99.5%.

2.3 Dispersing agent

Sodium dodecyl sulfate of purity 98% purchased from Sigma Aldrich.

2.4 Effervesence agents

Sodium bicarbonate anhydrous, free flowing with water solubility 100 mg/ml purchased from sigma Aldrich, Citric acid anhydrous (99.5%) , white crystalline powder, free flowing procured from Merck.,Tartaric acid purchased from sigma Aldrich.

2.5 Binder

Magnesium stearate technical grade purchased from sigma Aldrich.

2.6 Filler

Silica (Grade –Supersil 100) purchased from supersil chemical Pvt. Ltd. It is obtained in pure white amorpous powder, particle size 18–22 µm, Surface Area (110–125 M2 / gm), Moisture content at 105 °C for 1 hr is 5%,Bulk density- (0.08–0.11 gm/cc), (Oil absorption 240–270%)

All materials were used as purchased no further purification was required.

The bioefficacy evauation was done in Aedes agyptii in Institute of Pesticide Formulation Technology, Gurugram.

3.1 Preparation of tablet in three steps

3.1.3

3.1.3 Compression

All the homogeneously blended powdered composition was granulated by adding 1–2 g methanol in the homogenizer at speed of 200 rpm for 5 min. After granulation then granulated powdered material was transferred to tablet machine (AJANTA, Model Number/Name: Aei-tm-sh-13) funnel and compressed by tablet punch machine with 17.8 mm punch set (Fig. 1).

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Fig. 1

Flow diagram showing the sequence of tablet processing to make effervescence tablet.

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3.2 Precompression test

3.2.2

3.2.2 Flowability

Flowability was determined as per Collaborative International Pesticides Analytical Council (CIPAC MT-193). In this method powdered mixer with all active and inert ingredients were poured through the funnel. The mixed powder was poured through the funnel until it touches the tip of funnel. R is the radius of the conical pile and H is the hight of powdered cone as shown in Fig. 2. Flow ability was calculated by the formula described in eq.1 as given below:-

(1)

Tan α = H/R

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Fig. 2

Diagramatic representation of flowability measurement.

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3.3 Apparent bulk density

Apparent bulk density was measured by volume and weight measurement of the tablet powdered mixer. For apparent bulk density measurement, tablet mixer was weighed first and then pours in the measuring cylinder and packed without any tapping. The volume of the known amount of weight tablet mixer was noted from the graduated cylinder. Apparent density was calculated by following formula as described in eq (2):-

m = weight of powder (gms)

v = volume of tablet mixed (without tapping)

3.4 Packed bulk density

Same procedure was repeated as in apparent bulk density measurement. The only difference is that cylinder should be tapped till no change was seen in the volume mark of cylinder after putting powered content. Packed bulk density was calculated by following formula as described in Eq. (3):

m = weight of tablet mixer in gms for tight paking of cylinder

vb = volume of packed mixer (final tapped volume)

3.5 Compressibility percent (%)

Compressibility % was calculated by the putting obtained values of apparent bulk density and packed bulk density measurement as given in equation (4). The values were interpreted by the carr’s grading index as in Table 1.

3.6 Post compression analysis of tablet

3.7 Rate of disintegration

Disintergration is measured as per CIPAC method MT 197. In this method one effervessence tablet was put in 250 ml distilled water and mixed by glass rod by gentle stirring for specific time period. The absence of no residue left on sieve indicate complete disintergration of tablet in water. This test was repeated three times.

3.8 Moisture content

Ten tablets were weight and then dried in desicator with calcium sulphate for 4–6 hrs. Then the moisture content was calculated as follows:- $$\mathbf{M}\mathbf{o}\mathbf{i}\mathbf{s}\mathbf{t}\mathbf{u}\mathbf{r}\mathbf{e}\%=\frac{\mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\mathbf{o}\mathbf{f}\mathbf{t}\mathbf{a}\mathbf{b}\mathbf{l}\mathbf{e}\mathbf{t}\mathbf{b}\mathbf{e}\mathbf{f}\mathbf{o}\mathbf{r}\mathbf{e}\mathbf{d}\mathbf{r}\mathbf{y}\mathbf{i}\mathbf{n}\mathbf{g}-\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\mathbf{o}\mathbf{f}\mathbf{t}\mathbf{a}\mathbf{b}\mathbf{l}\mathbf{e}\mathbf{t}\mathbf{a}\mathbf{f}\mathbf{t}\mathbf{e}\mathbf{r}\mathbf{d}\mathbf{r}\mathbf{y}\mathbf{i}\mathbf{n}\mathbf{g}}{\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\mathbf{o}\mathbf{f}\mathbf{t}\mathbf{a}\mathbf{b}\mathbf{l}\mathbf{e}\mathbf{t}\mathbf{b}\mathbf{e}\mathbf{f}\mathbf{o}\mathbf{r}\mathbf{e}\mathbf{d}\mathbf{r}\mathbf{y}\mathbf{i}\mathbf{n}\mathbf{g}}\mathbf{x}100$$

3.9 Content analysis by UV– visible and HPLC

For content analysis twenty tablets were weighed and fine powder was made by grinding. An equivalent amount of 300 mg of neem oil dispersed silica was weighed and kept in 10 ml volumetric flask. The tablet powder was sonicated for 5 min and stir over magnetic stirrer for 10 min. After complete mixing then the mixer was filtered through Whatman cellulose filter paper and further aqeous dilutions were made. Finally, absorbance was measured at 208 nm by UV –visible spectroscopy(Perkin Elmer) against blank. Same filtrate was analyzed by HPLC (Shimadzu -(LC-2010AHT) by taking Azadirachtin as standard in following conditions:-

Stationary Phase – C-18 Column,

Mobile Phase –Acetonitrile/ Water (35:65).

Run Time-60 Minutes,

Flow Rate 1.2 ml/Min,

Injection Volume-10 µl,

Detector Wavelength −214

Volume of Injection-10 µl.FTIR

FT-IR was performed by Thermo fisher scientific ATR-FTIR(Model:Nicolet™) spectrophotometer. The FT-IR characterization was performed in crused powdered materials of tablet after post compression. The infrared (IR) Spectra were recorded and values are expressed as λ max cm⁻¹.

3.10 Accelerated stability test (ATS)

In ATS studies as per CIPAC method MT 46.3 in this method test tablet was stored in triplicates at 54 °C and room temperature for 15 days. After 15 days all the parameters were studied again for assessing the stability of effervescence tablet.

3.11 Bio-efficacy assay of neem effervescence tablet on Aedes aegypti

1. Maintenance of the mosquito larvae of Aedes aegypti

Bioassay of Aedes aegypti was maintained in Institute of Pesticide Formulation Technology,Gurugram,Haryana in standard conditions i.e 25 ± 5 ͦ C temperature and 70 ± 2% RH. The larvae were kept in water basins along with starch and cellulose as larval feed.

1. Larval bioefficacy

After assessment of physicochemical parameters effervessence tablet biefficacy was checked against larvae of mosquito species Aedes aegypti. The bioefficacy of tablet was measured in mortality percentage of larvae after application of tablet in 7 replicates. Larvicidal mortality test of the tablet was evaluated on 3rd instar larvae of Aedes aegypti as per WHO guidelines. Ten larvae was collected by the dropper and transferred into 250 ml containg dispersed tablet in water. Bio-assay was done in 7 replicates.

The experiment was carried out in seven different beakers as 7 replicates. Ten larvae was kept as control in separate beaker without tablet. Control and treated set was kept undisturbed at 25 ± 5 °C and 70 ± 8 RH. Mortality % was checked after 24 hr and 48 hr in replicates and control (Schnaubelt (1999) and then % mortality was calculated by abborts formula (Abbott 1925).

% Test mortality -% Control mortality × 100

% Mortality =

100 - % Control mortality

1. Ovipositional deterrent activity bioassay

The Ovipositional bioassay was carried out according to the method mentioned in (Xue et al. (2003)) with small modification. Twenty gravid females of Ae. aegypti female mosquitoes were released in iron framed mosquito cage having dimensions 30 × 30 × 25 cm. After 24hr the eggs were counted and ovipositional deterrent was calculated as ovipositional detterent percentage (%) calculation as shown below:-

Ovipositional Detterent Percentage (%) = E_C – E_T / E_C

E_C = No. of eggs laid in Control

E_T = No. of eggs laid in test (with dispersed tablet)

1. Ovicidal activity

Ovicidal activity was checked by mentioned method with slight modification (Su and mulla, 1998). The ovicidal test was carried out in 28 ± 1ͦC and 35–45% RH. In ovicidal test 25 gravid females were placed in the mosquito cage. Five bowls filled with 100 ml distilled water and one tablet was dispersed in each bowl. One bowl was filled with water and left without dispersing the tablet and treated as control. The hatching of eggs was checked after 40 hrs of oviposition and calculates the hatching rate.

% of egg mortality = Total no. of hatched larvae/total no. of eggs X 100

6.1 Optimized composition of effervescence tablet

All the inert ingredients and effervescence combination amount were optimized in 30% Neem oil along with wetting and dispersing agents. The effervescence combination was optimized separately. In effervescence tablets both the combinations were mixed homogenously and followed by compression in Table 2.

6.2 Mechanism of fast dispersion in water

Effervescence liberate CO₂ when dispersed in water by following chemical reaction (H. Stahl 2003):

1. Chemical reaction of citric acid with sodium bicarbonate is defined as:

C₆H₈O₇ (aq) + 3NaHCO₃ (aq) → Na₃C₆H₅O₇ (aq) + 3H₂O + 3CO2 (g) ↑

Citric acid + Sodium bicarbonate → Sodium citrate + Water + Carbon dioxide

1. Similarly Chemical reaction of tartaric acid with sodium bicarbonate is defined as:

H₂C₄H₄O₆(aq) + 2NaHCO₃ (aq) → Na₂C₄H₄O₆ (aq) + 2H₂O + 2CO₂ (g) ↑

Tartaric acid + Sodium bicarbonate → Sodium tartarate + Water + Carbon dioxide

6.3 Precompression test

6.4 Post compression analysis of tablet

6.4.9

6.4.9 Ftir

Neem oil effervesence tablet shows absorbance band at 3255.17 cm⁻¹ (Pyrollidine N-H Stretching) in PVP K-30 which act as dispersing agent, methy ester bands at (2952 cm⁻¹), C = O carboxylic acid of fatty acids (1744 cm⁻¹) in neem oil, C = C of aromatic ring at (1433 cm-1) in active ingredients as shown in Fig. 3. All the red arrows shows bands of neem oil and green arrows are corresponds to inert ingredients structural groups. FT-IR of effervessence tablet showed separate peaks for inert and active ingredients. Hence, the FT-IR data illustrates that tablet inert ingredient are nonreactive to neem oil and maintain the active constituents same as in neem oil in the tablet formulation.

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Fig. 3

FTIR analysis of effervescence tablet.

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6.5 Accelerated stability test

Accelerated stability testing is the validated method for predicting the stability of product for long period of time under different conditions. This test has significant role in setting shelf life of the any formulated product as Table 3.

The results of table 3 illustrated that after storing at various conditions tablet physicochemical evaluation data was slightly changed which was remain in the limits. This shows that tablet is stable during extended period of time at various conditions with same efficiency as freshly prepared tablet. It confirmed that no degradation and change was occurred during storage

6.6 Larval bioefficacy

There are many problems are associated with the use of synthetic pesticide for combating the mosquito problem like pesticide resistance, toxicity and many other harmful effects towards non-targets. Hence, there is urgent need for safe alternates like plant derived products which are safe and effective against mosquito control. The present study of Neem effervescence tablet gave on an average 85% larval mortality after dispersion in water and effective up to 1 week after application. Table shows that decrease in mortality percent was slightly varied from the initial this shows that the formulation has residual effect for long period of time. Neem has antifeeding property by altering the insects chemo receptors for food (Mordue (Luntz) AJ, Blackwell A, (1993)). Mosquito larvae when ingest the dispersed neem content which results larval mortality. Neem has growth inhibitor activity which will stop the larval growth and results into larval mortality(Nathan et al., 2005).The bio-activity was good due to their uniform dispersion in water and effectively control the mosquito larvae on breeding places.

Effervescence tablet gave on an average 85–90% larval mortality after 24 hrs and remain effective for 1 week with on an average 75% larval mortality after dispersion of four tablet in 250 ml water and effective up to 1 week after application. The neem tablet was compared with commercial grade larvicidal product. The larval mortality % was found more in developed formulation than commercial grade. The Table 4 shows, reduction in mortality percent was slightly varied from the initial. This tablet effectively controls the mosquito larvae on breeding places. The tablet is very effective for Instantaneous mosquito control problem. (See Table 5.)

6.7 Ovicidal activity

Ovicidal activity was found in neem effervescence tablets after 24 hrs of application 90–95% eggs were died and eggs were not hatched. Left 5–10% eggs hatched into feeble larval stages and not capable to set out pupae stage this indicate the growth inhibitory activity of the neem constituents in neem oil tablet. The ovicidal activity of neem effervescence tablet mainly due to the presence of bioactive constituent azadirachtin (A. indica) in the mosquito

eggs (Su and mulla, 1998). This means only one application is enough for termination of all early stages of mosquito as shown in table V in three replicates.

6.8 Ovipositional detterent

The effervescence tablet containing neem oil shows ovipositional detterent activity shown in table 6. The active ingredient of neem oil is azadirachtin content, many research studies identified it as ovicidal detterent in coleoptra, dipteral and Lepidoptera (Schmutterer, 1988).

The neem containing effervescence tablet gives maximum deterrent action in three dispersed tablet in 500 ml i.e. 80–90% ovipositional deterrent activity. Remaining laid eggs were not viable to hatch due to presence of azadirachtin content in water and results into egg mortality. The effectiveness of efferevesence tablet is upto 15 days. Reduction in egg laying over water surface will terminate the mosquito problem by targeted destruction of early cause of mosquito problem from the root level (Halder et al. (2011)).

6.9 Dissolved oxygen content of applied effervescence tablet aqueous medium

Dissolved oxygen is the main water component which regulate the survival of aquatic organisms in water (Alabaster, 1982). In respiration, an aquatic organism takes water oxygen by gills and some by skin and diffuses throughout the bloodstreams. Any declination in external dissolved oxygen causes several alterations in physiological as well as behavioral responses and ultimately results into death of fishes or some other permanent defects. Dissolved oxygen content of Neem effervesce tablet dispersed water was checked in lab conditions and local pond and compared with pure water dissolved oxygen content. Dissolved oxygen of original water was7.5 mg/l after dispersing effervescence tablet it was slightly reduced to 6.9 mg/l but within the limits of range of tolerance i.e 1–20 mg/l. This study indicates that after dispersion of effervesce tablet in aquatic bodies survival of non target organisms remain unaffected and water quality in terms of dissolved oxygen maintained.