Sci Pharm. 2008; 76: 269–277
doi:10.3797/scipharm.0803-14
© Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria
Reproduction is permitted for non-commercial purposes.
269
Preservative Evaluation of Novel 2,4-Hexadienoic Acid
Derivatives in Aluminium Hydroxide Gel – USP
Vikramjeet JUDGE 1, Balasubramanian NARASIMHAN * 1,
Rakesh NARANG 1, Ruchita OHLAN 2, Sucheta OHLAN 2
1
Department of Pharmaceutical Sciences, Guru Jambheshwar
University of Science and Technology, Hisar-125 001, Haryana
2
Hindu College of Pharmacy, Sonepat-131001, Haryana
Abstract
The potential derivatives of sorbic acid (2,4-hexadienoic acid) from our
previous study were subjected to preservative efficacy testing. Aluminium
Hydroxide Gel – USP was used as a pharmaceutical product and Staphylococcus
aureus MTCC 2901, Bacillus subtilis MTCC 2063, Escherichia coli MTCC 1652,
Candida albicans MTCC 227 and Aspergillus niger MTCC 8189 were used as
representative challenging microorganisms for antimicrobial effectiveness testing as
per USP 2004. The hexadienoic acid derivative, m-nitrosorbanilide has better
preservative efficacy than sorbic acid as well as the standard preservatives, methyl
paraben and propyl paraben.
Keywords
Sorbic acid • m-Nitrosorbanilide • 8-Quinolinyl sorbate • Preservative • Log CFU/mL
Introduction
Non-sterile pharmaceutical products with a high degree of water availability
may be contaminated with microorganisms. The contaminating microorganisms
may cause spoilage of the product with loss of therapeutic properties and, if they
are pathogenic, serious infections can arise [1]. During past 20 years, the frequency
of systemic infection has increased dramatically along with the number of invasive,
* Corresponding author: Tel.: +91-1662-263162; Fax: +91-1662-267240.
E-mail: [email protected] (B. Narasimhan).
270
V. Judge et al.:
mostly opportunistic, fungal species carrying infectious diseases. Fungal infections
are important cause of morbidity and mortality in hospitalized patients: candidiasis
is the fourth most common blood culture isolates at US hospitals, pulmonary
aspergillosis is the leading cause of death in bone marrow transplant recipients,
and Pneumocystis carinii pneumonia is the leading cause of death in AIDS patients
in North America and Europe [2]. To inhibit the growth of contaminating microorganisms, antimicrobial preservatives are introduced into the product during
manufacturing process for safety of products throughout its use by consumers [3].
In view of above, in the present study we have decided to search for some better
preservatives among potential antimicrobial compounds from a series of sorbic acid
(2,4-hexadienoic acid) derivatives recently reported by us [4].
Experimental
Materials
Nutrient agar, nutrient broth, sabouraud dextrose agar and sabouraud
dextrose broth were obtained from Himedia, Mumbai. Mannitol, methyl and propyl
paraben were obtained from CDH, Mumbai.
Methods
Aluminium Hydroxide Gel USP was used as the pharmaceutical product for
evaluation of preservative efficacy testing.
Preparation of Aluminum Hydroxide Gel-USP [5]
Formula
Aluminium hydroxide gel – 36 g; Mannitol – 7 g; Methyl paraben – 0.2 g;
Propyl paraben – 0.02 g; Saccharin – 0.05 g; Peppermint oil – 0.005 mL; Alcohol –
1 mL; Purified water q.s. – 100 mL
The weighed quantity of aluminum hydroxide gel and mannitol were triturated
with 50 mL of water in a mortar. Methyl paraben, propyl paraben, saccharin and
peppermint oil were dissolved in alcohol and added to above mixture and triturated
well. The volume was made up to 100 mL with purified water.
Preservative Evaluation of Novel 2,4-Hexadienoic Acid Derivatives …
271
For preservative efficacy testing, the aluminium hydroxide gel was prepared
using the preservatives mentioned in Table 1 by replacing methyl paraben and
propyl paraben from the above formula. The equimolar amount of selected
preservatives (Table 1) were calculated with reference to the amount of methyl
paraben (0.0013 mol) and added into the pharmaceutical products.
Tab. 1. Amount of selected preservatives added in the pharmaceutical products
Code Preservative
Amount (% w/w)
P1
Sorbic acid
0.15
P2
8-Quinolinylsorbate
0.31
P3
m-Nitrosorbanilide
0.30
Preservative efficacy testing in pharmaceutical products [6]
Aluminum hydroxide gel prepared with different preservatives was sterilized in
autoclave at 1200C for 15 minutes. The products were then inoculated separately
with 2 x 104 CFU/mL of Staphylococcus aureus MTCC 2901, Bacillus subtilis MTCC
2063, Escherichia coli MTCC 1652, Candida albicans MTCC 227 and Aspergillus
niger MTCC 8189 and stored at room temperature (250C). The CFU/mL of the
product was determined at an interval of 0, 7, 14, 21 and 28 days. The experiment
was performed in triplicate. The log values of number of colonies of microorganisms
per ml along with their log standard deviation values (Table 2 – Table 6) in
aluminium hydroxide gel was calculated and compared as per the guidelines of
USP 2004.
Results and Discussion
For B. Subtilis:
The log results are shown in Table 2. The parent compound sorbic acid was
effective upto the limit prescribed by USP on 14th day (0.000 ± 0.00) but it could not
pass the limit on 28th day (0.699 ± 0.04). The derivatives m-Nitrosorbanilide and 8Quinolinylsorbate were found to be effective on 14th day (0.477 ± 0.09, 0.000 ± 0.17
272
V. Judge et al.:
respectively) and 28th day (0.477 ± 0.08, 0.301 ± 0.08 respectively) as the log
results were found to be in accordance with limit prescribed in the USP. The
standard preservative was active on 14th day (0.000 ± 0.00) but fails to meet the
required limit on 28th day (0.778 ± 0.03).
Tab. 2. Bacterial count (CFU/mL) of B. subtilis in Aluminium Hydroxide Gel USP
supplemented with preservatives
Compound
Log (CFU/mL) ± SD (Time in days)
0
Sorbic acid (P1)
0.00
0.00
0.00
0.17
0.00
0.00
0.09
0.17
0.602 ± 0.477 ± 0.000 ± 0.000 ±
0.05
Control
0.17
0.301 ± 0.000 ± 0.477 ± 0.000 ±
0.08
Methyl and Propyl paraben
21
0.000 ± 0.000 ± 0.000 ± 0.000 ±
0.17
m-Nitrosorbanilide (P3)
14
0.000 ± 0.000 ± 0.000 ± 0.000 ±
0.00
8-Quinolinyl sorbate (P2)
7
0.09
0.00
0.00
0.699 ± 0.602 ± 1.110 ± 0.301 ±
0.04
0.05
0.02
0.08
28
0.699 ±
0.04
0.301 ±
0.08
0.477 ±
0.08
0.778 ±
0.03
0.845 ±
0.03
For S. aureus:
As per the results given in Table 3 sorbic acid was found to be active against
S. aureus on 14th (0.000 ± 0.17) as well as 28th (0.602 ± 0.05) day. The derivatives
m-Nitrosorbanilide (0.000 ± 0.17, 0.301 ± 0.08) and 8-Quinolinyl sorbate (0.000 ±
0.00, 0.000 ± 0.00) showed results better than sorbic acid and are within the
pharmacopoeial limits. The standard methyl paraben (0.000 ± 0.00, 0.477 ± 0.09)
also passes the prescribed limits.
Preservative Evaluation of Novel 2,4-Hexadienoic Acid Derivatives …
273
For E. coli:
Sorbic acid (0.000 ± 0.17 (14th day), 0.477 ± 0.09 (28th day)) meets the
pharmacopoeial limits and m-Nitrosorbanilide (0.000 ± 0.17 (14th day), 0.301 ± 0.08
(28th day)) showed results better than sorbic acid and meets the required limits.
8-Quinolinyl sorbate exceeds the pharmacopoeial limits on 14th day (0.602 ± 0.05)
by marginal difference but passes the limit on 28th day (0.000 ± 0.08) as complete
microbial inhibition was seen at that period. The standard (0.602 ± 0.05 (14th day),
0.699 ± 0.04 (28th day)) fails to meet the limits. The results are shown in Table 4.
Tab. 3. Bacterial count (CFU/mL) of S. aureus in Aluminium Hydroxide Gel USP
supplemented with preservatives
Comp.
Log (CFU/mL) ± SD (Time in days)
0
Sorbic acid (P1)
Methyl and Propyl paraben
Control
21
0.00
0.17
0.04
0.301 ± 0.000 ± 0.000 ± 0.000 ±
0.08
m-Nitrosorbanilide (P3)
14
0.301 ± 0.000 ± 0.000 ± 0.699 ±
0.08
8-Quinolinyl sorbate (P2)
7
0.17
0.00
0.00
0.602 ± 0.000 ± 0.000 ± 0.000 ±
0.05
0.00
0.602 ±
0301 ±
0.05
0.08
0.17
0.00
0.000 ± 0.301 ±
0.00
0.08
0.903 ± 0.477 ± 0.602 ± 0.778 ±
0.02
0.08
0.05
0.03
28
0.602 ±
0.05
0.000 ±
0.00
0.301 ±
0.08
0.477 ±
0.09
0.778 ±
0.03
For C. albicans:
Sorbic acid (0.000 ± 0.00 (14th day), 0.000 ± 0.00 (28th day)) passes the limit of
preservative efficacy test and the derivatives m-Nitrosorbanilide (0.000 ± 0.00 (14th
day), 0.000 ± 0.00 (28th day)) and 8-Quinolinyl sorbate (0.301 ± 0.08 (14th day),
274
V. Judge et al.:
0.000 ± 0.00 (28th day)) were found to be active in accordance with USP. The
standard (0.602 ± 0.05 (14th day), 0.000 ± 0.00 (28th day)) also meets the limits and
the test compounds showed results better than standard. The results are presented
in Table 5.
Tab. 4. Bacterial count (CFU/mL) of E. coli in Aluminium Hydroxide Gel USP
supplemented with preservatives
Comp.
Log (CFU/mL) ± SD (Time in days)
0
Sorbic acid (P1)
0.17
0.08
0.17
0.05
0.08
0.00
0.17
0.08
0.778 ± 0.000 ± 0.602 ± 0.301 ±
0.03
Control
0.00
0.699 ± 0.000 ± 0.000 ± 0.301 ±
0.04
Methyl and Propyl paraben
21
0.000 ± 0.000 ± 0.602 ± 0.301 ±
0.00
m-Nitrosorbanilide (P3)
14
0.301 ± 0.000 ± 0.000 ± 0.301 ±
0.08
8-Quinolinyl sorbate (P2)
7
0.00
0.05
0.08
0.845 ± 0.602 ± 0.778 ± 0.954 ±
0.03
0.05
0.03
0.02
28
0.477 ±
0.09
0.000 ±
0.00
0.301 ±
0.08
0.699 ±
0.04
1.041 ±
0.05
For A. niger:
Sorbic acid (0.903 ± 0.02 (14th day), 0.000 ± 0.00 (28th day)),
m-Nitrosorbanilide (0.477 ± 0.09 (14th day), 0.000 ± 0.00 (28th day)) and 8Quinolinylsorbate (0.602 ± 0.05 (14th day), 0.602 ± 0.05 (28th day)) were found to be
active against the fungus. The test compounds m-Nitrosorbanilide and 8-Quinolinyl
sorbate were more active than sorbic acid and comparable to standard methyl
paraben (0.699 ± 0.04 (14th day), 0.477 ± 0.08 (28th day)). The results are presented
in Table 6.
Preservative Evaluation of Novel 2,4-Hexadienoic Acid Derivatives …
275
Tab. 5. Fungal count (CFU/mL) of C. albicans in Aluminium Hydroxide Gel USP
supplemented with preservatives
Comp.
Log (CFU/mL) ± SD (Time in days)
0
Sorbic acid (P1)
7
0.00
0.08
0.00
0.00
0.04
0.05
0.03
0.03
0.000 ±
0.00
0.000 ±
0.17
0.00
0.000 ±
0.17
0.00
0.000 ±
0.03
0.477 ± 0.778 ± 0.845 ± 0.845 ±
0.08
28
0.00
0.301 ± 0.699 ± 0.602 ± 0.778 ±
0.08
Control
0.00
0.000 ± 0.000 ± 0.000 ± 0.000 ±
0.17
Methyl and Propyl paraben
0.17
0.301 ± 0.000 ± 0.301 ± 0.000 ±
0.08
m-Nitrosorbanilide (P3)
21
0.301 ± 0.000 ± 0.000 ± 0.000 ±
0.08
8-Quinolinyl sorbate (P2)
14
0.00
0.903 ±
0.03
0.02
Tab. 6. Fungal count (CFU/mL) of A. niger in Aluminium Hydroxide Gel USP
supplemented with preservatives
Comp.
Log (CFU/mL) ± SD (Time in days)
0
Sorbic acid (P1)
0.04
0.02
0.17
0.00
0.08
0.05
0.09
0.05
0.04
0.09
0.08
0.00
0.301 ± 0.301 ± 0.699 ± 0.000 ± 0.477 ±
0.08
Control
28
0.301 ± 0.699 ± 0.477 ± 0.301 ± 0.000 ±
0.08
Methyl and Propyl Paraben
21
0.602 ± 0.477 ± 0.602 ± 0.477 ± 0.602 ±
0.05
m-Nitrosorbanilide (P3)
14
0.477 ± 0.699 ± 0.903 ± 0.000 ± 0.000 ±
0.09
8-Quinolinyl sorbate (P2)
7
0.08
0.04
0.00
0.08
0.699 ± 1.079 ± 0.954 ± 1.000 ± 1.079 ±
0.04
0.01
0.02
0.00
0.02
276
V. Judge et al.:
The test compound m-Nitrosorbanilide (P3) was active against all the tested
microbial strains under the standard test conditions as per USP NF 2004. It has
shown potential to be chosen as a pharmaceutical preservative. 8-Quinolinyl
sorbate also had shown good results except against E. coli against which it was
active only in later period of testing interval.
Conclusion
The derivatives selected for preservative efficacy testing have shown
promising results. The test compound m-Nitrosorbanilide (P3) was active against all
the tested microbial strains under the prescribed test conditions as per the criteria
of USP 2004 i.e for antacid made with an aqueous base, preservative effectiveness
are met if there is no increase from initial calculated count at 14th and 28th days in
case of bacteria, yeast and moulds. Where no increase is defined as not more than
0.5 log10 higher than previous value measured. The above fact was supported by
the log CFU/mL values of m-nitrosorbanilide (P3) for 0 – 28 days viz. 0.301 – 0.447
(S. aureus), 0.602 – 0.301 (B. subtilis), 0.699 – 0.301 (E. coli), 0.000 – 0.000 (C.
albicans), 0.301 – 0.000 (A. niger) which were according to the prescribed USP
criteria. The results of preservative efficacy testing indicated that m-nitrosorbanilide
(P3) has the potential to be chosen as a pharmaceutical preservative.
References
[1]
Zani F, Minutello A, Maggi L, Santi P, Mazza P.
Evaluation of preservative effectiveness in pharmaceutical products: the use
of a wild strain of Pseudomonas cepacia.
J Appl Microbiol. 1997; 83: 322–326.
[doi:10.1046/j.1365-2672.1997.00231.x]
[2]
Garcia-Domenech R, Rios-Santamariana I, Catala A, Calabuig C, Castillo
LD, Galvez J.
Application of molecular topology to the prediction of antifungal activity for a
set of dication-substituted carbazoles, furans and benzimidazoles.
J Mol Str. 2003; 624: 97–107.
[doi:10.1016/S0166-1280(02)00740-6]
Preservative Evaluation of Novel 2,4-Hexadienoic Acid Derivatives …
277
[3]
Denyer SP, King RO.
In Microbial Quality Assurance in Pharmaceuticals, Cosmetics and Toiletries
Ed. Bloomfield SF, Baird R, Leak RE, Leech R, Chichester: Ellis Horwood:
1988: 156–170.
[4]
Narasimhan B, Judge V, Narang R, Ohlan R, Ohlan S.
Quantitative Structure Activity Relationship Studies For Prediction of
Antimicrobial Activity of Synthesized 2,4-Hexadienoic Acid Derivatives.
Bioorg Med Chem Lett. 2007; 21: 5836–5845.
[doi:10.1016/j.bmcl.2007.08.037]
[5]
Lachman L, Lieberman HA, Kanig JL.
In: The Theory and Practice of Industrial Pharmacy
3rd Ed., Vargheese Publishing House, Bombay, 1987: 498–499.
[6]
The United States Pharmacopoeia.
United States Pharmacopoeial Convention, Inc, Rockville, 2004: 2148–2150.
Received March 18th, 2008
Accepted (after revision) May 6th, 2008
Available online at www.scipharm.at May 22nd, 2008