Formulation of Ointment with Protein Extract

MITITELU Magdalena1, PRISADA Răzvan Mihai2*, NICOLESCU Teodor Octavian3, NEACȘU Sorinel Marius1, NICOLESCU Florica4, IONIȚĂ Ana Corina1, POLL Alexandru5, DUMITRESCU Denisa Elena6

1Clinical Laboratory and Food Hygiene Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

2Physical and Colloidal Chemistry Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

3 Organic Chemistry Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

4 Toxicology Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

5Anatomy Department, Faculty of Dentistry, ”Carol Davila” University of Medicine and Pharmacy, 17-23, Calea Plevnei, 060015, Bucharest (ROMANIA)

6 Organic Chemistry Department, Faculty of Pharmacy, Ovidius University, Constanța (ROMANIA)

*corresponding author: razvanprisada@yahoo.com

Abstract

    The aim of this work is to prepare and analyse a protein extract from mussels (Mytilus galloprovincialis) with biological activity. The protein extract resulted by boiling mussels in two stages was concentrated by counter-dialysis with polyvinylpyrolidone and then lyophilised. The total extract lyophilised has manifested a bacteriostatic action on a culture medium of Escherichia coli. It was prepared an ointment with the extract. The product resulted was analysed according to the present standards. The results have shown a good stability for the ointment. 

Keywords: protein extract, mussels, ointment

Introduction

     The marine ecosystem is part of the largest aquatic system on the planet, covering about 70% of the Earth’s surface. The diversity and productivity of marine ecosystems are very important in maintaining the health of the marine and terrestrial environment and provides important sources of food for humans and animals, for the pharmaceutical, food and cosmetics industries, etc [1,2,3]. Literature of The specialty is rich in presenting medicines from marine resources, but it is modest in what concerns the use of marine resources in the Black Sea in the pharmaceutical field [4-8]. 

     The quality of the marine environment influences the composition of marine resources, so that intense pollution causes the increase of contaminants in marine life [9-13].

     Seafood includes a wide variety of mollusks (snails, mussels, oysters, shells, squid, octopuses) and crustaceans (crayfish, lobsters, crabs, shrimp). Seafood is part of the daily diet of Asian and Mediterranean populations, and can be eaten both raw and prepared, and in recent years have begun to be more frequently consumed in Romania [14,15,16]. The mussels are very appreciated by the nutritionists for their complex composition.  Their meat is known for his nutritive value give it by the presence of all vital components for human food: proteins, essential amino acids, lipids, glucides, macro and micro-elements, enzymes, vitamins etc. [17-22]. 

      The purpose of the present work is to obtain a mussel extract with biological activity for pharmaceutical practice.

Methodology

     The Mytilus galloprovincialis specimens longer than 5-6 cm were collected off the Romanian coast at Agigea and Eforie. 

     The mussel extract was obtained in two stages. In the first stage we extracted 2.5 kg of mussels with shells in 2.5 L of water by boiling at 100 0C for 60 min. After cooling, the liquid fraction was separated by decantation. We have obtained 3.5 ± 0.1 L liquid fraction and 1.5 ± 0.2 kg of mussels.

     Then, the meat was separated by shells manually and we have obtained 0,3 ± 0,05 kg of meat and 1.2 ± 0.2 kg of shells. The separated meat followed the second stage of extraction. The mussel meat and the liquid fraction resulted from the first extraction were further extracted at 100 0C for 30 min. After cooling, the liquid fraction separated by decantation from the oily fraction was filtered under vacuum through Buchner funnel. 

      The resulted solution was concentrated by counter-dialysis with polyvinylpyrolidone and then lyophilised. 

       In order to characterise the obtained extract were made following determinations: total proteins by Lowry method [21], total lipids after Christie [22], and total glucides by method with orcinol and the loss on drying. We have determined the electrical conductibility and pH on watery solution 0.1% using Radelkis pH-meter-coductometter and spectral characteristics with UV-VIS Cecil 2501 spectrophotometer. We have also analysed the action of the lyophilised extract on a culture of Escherichia coli. The strain was shown on nutritive gloze with following composition (g%): meat extract 0.3; peptone 1; NaCl 0.5; agar 1.8; distilled water 100 mL; pH = 7.4 – 7.6. On medium surfaces solutions of phenol (1%; 0.5%; 0.3%; 0.1%) and watery solution of lyophilised total extract of mussels 10%were added drop by drop. The bacteriostatic action of mussel extract was determined after incubation at 37 0C using the estimation of the inhibition zone size in report with solutions of phenol. The determinations were made after 24 and 48 hours.

       The protein extract was used for prepare an ointment with the composition presented in Table 1.

Table 1. The ointment with protein extract composition

Component Formula (g)
Protein extract 2
Simple ointment 92
Conserving solution 5
Lemon oil

1

     The ointment was prepared by mixing the melted simple ointment with mussel extract dissolved in conserving solution. The mixture was stirred until cooling. Finaly we have added volatile lemon oil. The preservative solution, which contains methyl paraben and propyl paraben (3:1 w/w) in concentration of 0.1%. The simple ointment is an official ointment base from Romanian Pharmacopoeia X which contains lanoline and vaseline [25]. 

      The ointment with 2% extract was submitted at some control tests in order to determinate its main characteristics and stability:

  • the determination of the appearance was made by examination with the magnifying glass (4.5x) of a sample stretched by thin layer on microscopic blade;
  • the determination of the main characteristics (odor and color) was made according to Romanian Pharmacopoeia X [25];
  • the determination of the emulsion type was made by following tests: dilution test, color test and electrical conductibility test [26,27];
  • the pH was determined after a preliminary manufacture of the ointment, respectively after the extraction of a sample with water (1:5) following by the measuring of the pH of watery phase, thus: at 5g sample were added 25 mL of distilled water and the mixture was stirred into an Erlenmeyer glass with cork heating on the water bath at 600C for 10 min. After cooling, the watery phase was separated by the oily phase and on watery phase the pH was determined with Radelkis pH-meter;
  • the determination of the viscosity was made with rotational Brookfield LFV viscometer [26,27];
  • the determination of dropping point was made with Ubbelohde apparatus [27];
  • the determination of the stability was made by maintaining the samples at two temperature conditions (20C and 400C): into a weighing ampoule with lid was introduced 5g of sample and the ampoule is maintained 8 hours at mentioned temperatures. Then, was examined the appearance of sample; 
  • the spreading capacity of the creams was determined by measuring the spreading diameter of 1 g of the ointment sample between two 20 x 20 cm glass plates after 1 min. The mass of the upper plate was standardized at 125 g. Weighs of 50 g, 100 g, 150 g, 250 g, 500 g and 750 g were subsequently placed over the sample at 1 min. intervals. The spreading areas reached by the sample were measured in millimeters in the vertical and the horizontal axes [27-30]. The results were expressed in terms of the spreading area as a function of the applied mass according to the following equation:

Si = di2 (π / 4)

in which Si is the spreading area (mm2) resulting from the applied mass i (g) and di2 is the mean diameter (mm) reached by the sample.

Results 

     The physical-chemical and biochemical characteristics of the lyophilised mussel extract are presented in Table 2.

Table 2. Characteristics of lyophilised mussel extract        

Characteristics Results
Appearance white-yellowish power, hygroscope
Solubility easy soluble in water, partially soluble in benzene (50.75%) and chloroform (28.51%), insoluble in acetone 
Total proteins (%) 34 – 38
Total glucides (%) 32 – 36
Total lipids absent
Electrical conductibility (solution 0,1%) 2 660 μS
pH (solution 0,1%) 7.8 – 8.2
λmax  258 – 260 nm
Loss on drying (%) 10 – 15
Mineral residue (at 5050C) 32-36 % (from dried substance)

     From the Table 2 we can observe that the extract is a glycoprotein product, water-soluble with a weak alkaline pH in solution. The analysis of the UV absorption spectra has emphasised a maximum absorption at 260 nm.

      In Table 3 are presented the results obtained from investigation of the lyophilised mussel extract activity on culture medium by measuring the inhibition zones.   

Table 3. Size of inhibition zones

Sample Inhibition zone (mm)

24h                               48h
Solution of phenol 1% 8.5                                 5
Solution of phenol 0.5% 5.5                                 3
Solution of phenol 0.3%   3                                   0.5
Solution of phenol 0.1%       1                                  absent
Solution of mussel extract 10%       3                                  absent

     The data from Table 3 show a bacteriostatic activity of the mussel extract comparable with a phenol solution of 0.3%.

      The characteristics of ointment prepared are presented in Table 4.       

                 Table 4. The ointment characteristics                         

Characteristics Results 
macroscopic characteristics appearance: homogenous;

colour: white-yellowish;

smell: specific
emulsion type water / oil (W/O)
pH 6.0 – 6.5
viscosity (cP)  325
dropping point (0C) 43

 

       The data from Table 4 show that the ointment is homogenous, with a pH appropriated with skin pH. Moreover, the preparation has presented a good stability by maintaining the homogenous appearance at 20C and 400C.

       The spreading capacities of the ointment with protein extract are presented in Fig. 1.

S (mm2)

Fig. 1  Spreading area function of the applied mass

We remark from Fig. 1 good values of spreading capacity.

Conclusions

     The obtaining of the watery extract by boiling present some advantages:

  • sterilisation of the material;
  • coagulation of some ballast substances which can be easy eliminated;
  • enrichment of the extract in active compounds. 

     The counter-dialysis was realised for to purify the extract by elimination of salts and small organic molecules. The efficiency of the dialysis regarding the elimination of salt charge was determined by the measurement of electrical conductibility.

     The protein mussel extract has presented a bacteriostatic activity on Escherichia coli comparable with a phenol solution of 0.3%.

     The ointment prepared with protein extract obtained from mussels has presented a good stability.

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