Stability Studies on the Aqueous Extract of the Fresh ...

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J. Chem. Chem. Eng. 6 (2012) 417-424

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Stability Studies on the Aqueous Extract of the Fresh Leaves of Combretum Micranthum G. Don Used as Antibacterial Agent Uduma Eke Osonwa1, Chukwuebuka Emmanuel Umeyor1*, Uduak Victor Okon2, Emmanuel Maduabuchi Uronnachi1 and Calistus Dozie Nwakile1 1. Department of Pharmaceutics/Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu 422001, Anambra State, Nigeria 2. Department of Pharmaceutics/Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Akwaibom State 520001, Nigeria

Received: April 04, 2012 / Accepted: May 08, 2012 / Published: May 25, 2012. Abstract: The fresh leaves of Combretum micranthum were harvested during the rainy season in Akwa Ibom State, washed and subjected to extraction by maceration in distilled water. Concentration was done with a rotary evaporator. The extracts were tested for content of the following phytochemicals using standard methods: alkaloids, saponins, tannins, anthraquinones, cardiac glycosides, flavonoids and steroids. Antibacterial sensitivity test of the extract was carried out on S. aureus, B. subtilis and E. coli using agar well diffusion method and Ampiclox® as control. The effect of storage time of the extract on its antibacterial activity was determined after the extract was kept at room temperature for 1 h, 24 h, 48 h and 72 h, respectively. Preparations of 200 mg/mL of the aqueous extract were made with additives as follows: (a) no additive; (b) methyl paraben (0.3%); (c) propyl paraben (0.3%); (d) sodium metabisulfite (0.01%); (e) A combination of methyl paraben (0.3%) and propyl paraben (0.3%); (f) A combination of methyl paraben (0.3%), propyl paraben (0.3%) and sodium metabisulfite (0.01%). A visual observation of the stabilized preparations was employed on daily basis for 30 days. Phytochemical analyses on the aqueous extract of the fresh leaves showed the absence of alkaloids, anthraquinones and flavonoids. Saponins, cardiac glycosides and steroids were present in moderate amounts and there was abundance of tannins. The extract had similar effect on both S. aureus and E. coli cultures but activity was very low with B. subtilis. When S. aureus activity increased with time of storage up to 48 hours, thereafter the activity started reducing. However, the activity of E. coli continued to increase with time (within the study period). Thus, it appears reasonable to store the extract for at least 48 hours before the use for enhanced activity. Key words: Combretum micranthum, fresh leaves, aqueous extract, antibacterial, stability.

1. Introduction The generality of the man’s life is 100% dependent on the plants in the environment [1]. This means that the diversity of plant species in the world is so useful for the adaptation to the environment and existence. *Corresponding author: Chukwuebuka Emmanuel Umeyor, Master, research field: nanomedicine and drug delivery. E-mail: [email protected]; Tel: +2348063299850.

The use of plants as the source of remedies for the treatment of many diseases dates back to prehistory and people of all continents have this old tradition. Despite the remarkable progress in synthetic organic chemistry of the 20th century, over 25% of prescribed medicines in industrialized countries denied directly or indirectly from plants [2]. A medicinal plant is defined by the World Health Organization (WHO) consultative group as any plant in which one or more of its organs contains substances

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Stability Studies on the Aqueous Extract of the Fresh Leaves of Combretum Micranthum G. Don Used as Antibacterial Agent

that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs [3]. The medicinal plants with high therapeutic and prophylactic value are mostly from the plants that are herbaceous and the majority of the world’s population uses such herb products as a primary source of medicine [4]. However, plants used in traditional medicine are still understudied, particularly in clinical microbiology [5]. According to Sofowora [6], there are about 149 plants that are commonly used in traditional medicine in Africa. It is reported that most antibiotics derived from microorganisms, and one to three antibiotics are launched every year [7]. Infectious diseases which are caused by bacteria, viruses, fungi and other parasites are major causes of death in humans, in spite of the enormous progress recorded by the modern medicine. The WHO [8] and UNAIDS [9] reported that between 14 and 17 million people die each year due to infectious diseases. In developing countries where medicines are quite expensive, investigation on antimicrobial activities from ethnomedicinal plants may still be needed. It is obvious that these phytochemicals will find their way in the armamentarium of physicians [10]. Any antibiotic has a limited effective life and the public is becoming increasingly aware of problems with the over prescription of these antibiotics. In addition, many people principally in the developed countries are interested in having more autonomy over their medical care, so self-medication is commonplace [11]. In developing countries, notably in West Africa, new drugs are not often affordable. Thus, up to 80% of the populations use medicinal plants as remedies [12]. In Nigeria, a lot of the rural populace still relies much on herbs for their health care needs. Citrus aurantifolia has been used in various Nigerian homes because of its antimicrobial activities [13]. Reports in Refs. [14-15] indicate that traditional healers throughout Africa have confined themselves almost exclusively to the use of species from the genus Combretum and the Terminalia in the treatment of a wide range of diseases to a lesser

extent. Combretum species are used for many medicinal purposes such as diarrhoea, pneumonia, gonorrhea, syphilis, malaria, hypertension, and even cancer [16]. Combretaceae is a large family with at least 475 species in the world and 244 species in Tropical Africa. Some species of the Combretaceae family have been investigated and have revealed to possess a large number of bioactive compounds, and to exert strong antioxidant and antimicrobial activities. Among those plants are Combretum micranthum, Combretum erytrophyllum and Terminalia catappa [17-19]. Combretum micranthum G. Don is a bushy shrub or creeper that may grow up to 20 m in length. The leaves are opposite, ovate and acuminate; the flowers are borne as auxiliary cluster on scaly stalks; the fruits are small with scaly and four winged [20]. The medicine value of the plant is due to the presence of certain substances such as amino acids, phenanthrenes and dihydrophenanthrenes, gums and glycosides [21-24]. The present study is aimed to investigate the phytochemical profile, the antibacterial effects of Combretum micranthum and the effect of storage time of the extract on the above activity as well as to assess the stability profile of aqueous preparations of the extract.

2. Materials and Methods 2.1 Materials Distilled water and nutrient agar were obtained from Oxion Ltd in Basingstokes in Hampshire of England; sodium metabisulphite, methylparaben and propylparaben were from BDH Chemicals Ltd (Poole, England); petri dish was from Bibby Sterilin in UK; Ampiclox® was from Beecham Pharmaceuticals, (Brentford, England), microbial cells of Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC 10418) and Bacillus subtilis (NCTC 8853) were from Pharmaceutical Microbiology Laboratory of Faculty of Pharmacy of University of Uyo in Nigeria.


2.2 Harvesting and Identification of Plant Material The Fresh leaves of the plant were harvested in August in 2010 from a farmland at Afaha Oku in Uyo Local Government Area of Akwaibom State, Southern Nigeria where they grow naturally. The leaves were thoroughly washed with clean water and properly rinsed. They were identified at the Herbarium Unit of the Department of Pharmacognosy and Natural Medicine, Faculty of Pharmacy, University of Uyo. A voucher specimen was deposited in the herbarium. 2.3 Extraction of Plant Material The Fresh leaves were chopped to small pieces, weighed and then pulverized in the laboratory. Exactly 100 g of the pulverized leaves was completely soaked in 500 mL of distilled water at room temperature for 48 h. After 48 h, the extract was sieved using a funnel which was fitted with cotton wool as sieve, then the filtration used No. 1 Whatman filter paper and the filtrate was collected in a beaker. The filtrate was concentrated to dryness using a rotary evaporator with water bath set at 40 °C until a dry and dark-green extract was obtained. The extract was properly stored in an air-tight container for further studies. 2.4 Yield of Extract The percentage yield of the extract was obtained after extraction using Eq. (1): W (1) Percentage Yield (%) = a  100 Wb where: Wa = Weight of obtained extract; Wb = Weight of extracted leaves. 2.5 Phytochemical Screening The extract was tested for the presence of different phytochemicals: alkaloids, saponins, tannins, anthraquinone, cardiac glycosides, flavonoids and steroids, which used the standard methods prescribed in Trease and Evans [25]. 2.6 Antibacterial Sensitivity Tests The Agar-well diffusion method was used to

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evaluate the antibacterial activity. Zones of inhibition of the extract were determined. 2 mg of the extract was freshly diluted using distilled water to obtain a concentration of 200 mg/mL, and used immediately for the test. Sterile petri dishes (diameter = 10 cm) were prepared with a base layer of nutrient agar and allowed to solidify. Bacteria cells at the density of 106-108 cfu were inoculated on the solid agar. Wells of 5 mm were made in the agar with a sterile cork borer and filled with 0.1 mL of the 200 mg/mL concentration of the extract. Also, 0.1 mL of 1 mg/mL concentration of Ampiclox® (standard antibiotic) was used as the positive control. Petri dishes were incubated at 37 oC for 24 h after standing for 2 h at room temperature to allow for diffusion. The diameters of clear zones which are indicative of inhibition of the organisms by the extract were measured in millimeters, as demonstrated by Abayomi [26]. The experiments were carried out in triplicates. The procedure was repeated with B. subtilis and E. coli as test organisms. 2.7 Effect of Storage Time of the Extract on the Antibacterial Activity The Effect of storage time of the extract on the antibacterial activity was determined by the above described agar-well diffusion method. A fresh dilution of the extract was prepared using distilled water to obtain a concentration of 200 mg/mL of the extract which was stored at room temperature for 1 h, 24 h, 48 h and 72 h respectively before the use for the bioassay. 2.8 Stability Testing of the Extract The ability of the extract to resist microbial growth and degradation on storage was assayed. This was carried out on the extract alone, and after the incorporation of some additives. The preparations were as follows:  Extract alone;  Extract + methyl paraben (0.3% v/v);  Extract + propyl paraben (0.3% v/v);  Extract + sodium metabisulphite (0.0% v/v);  Extract + methyl paraben (0.3% v/v) + propyl

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paraben (0.3% v/v);  Extract + methyl paraben (0.3%v/v) + propyl paraben (0.3% v/v) + sodium metabisulphite (0.01%v/v). Six preparations were visually observed daily for 30 days. The degree of clarity of the observed preparations was used to assess the stability of preparations. 2.9 Statistical Analysis The results from replicated experiments were expressed as mean ± standard deviation.

3. Result and Discussion 3.1 Yield of Extract The percentage yield of the extract which was obtained after aqueous extraction of the plant and calculated by Eq. (1) was 19.5%. This is appreciable considering that water used for the extraction is a polar solvent and will only extract polar constituents of the crushed plant leaves. 3.2 Phytochemical Screening The phytochemical screening which carried out on pulverized leaves gave the result, as shown in Table 1. In Table 1, alkaloids, anthraquinones and flavonoids were absent, while tannins, saponins, cardiac glycosides and steroids were present; saponins, cardiac glycosides and steroids were present in trace amounts while tannins were the major phytochemical present in the leaves extract. The presence of these metabolites in Table 1 Result of Phytochemical Screening of Extract of Combretum micranthum. Phytochemical Abundance Alkaloids Anthraquinones Flavonoids Tannins ++ Saponins + Cardiac glycosides + Steroids + -: Absent; +: Present in trace amount; ++: Present in appreciable amount.

some species of Combretum had already been reported [27-29]. High tannin content in leaves of Combretum micranthum had been reported [18]. However, many research works reported that the contents in metabolites, phenolics and flavonoid compounds as well are related to the nature of soil and microclimate [30]. The leaves of used Combretum micranthum for this research were harvested in August. Kana et al. [31] demonstrated that flavonoid contents increased from October to December, with the tendency to decrease from May to June. This could explain why there was absence of flavonoids in the extract. 3.3 Antibacterial Tests The antibacterial screening of the extract was done by the agar-well diffusion method. The mean inhibition zone diameter (IZD) which was measured was recorded in millimeter (mm). The obtained results are presented in Table 2. Table 2 showed that Ampiclox®, a broad spectrum antibiotic, exerted strong inhibitions against the tested strains of S. aureus, E. coli and B. subtilis. The result showed that the extract inhibited the growth of tested organisms at the test concentration of 200 mg/mL. It exhibited the highest and equal growth inhibition on S. aureus and E. coli with zones of inhibition values of 33.7 mm, but it produced the least growth inhibition on B. subtilis with zone inhibition value of 5.76 mm. This is in agreement with reports that many Combretum species have medical applications against many bacterial infections such as gonorrhea, syphilis and diarrhoea [16]. The high inhibition which was produced by the extract might be due to high amount of Table 2 Result of Antibacterial Screening of Extract of Combretum micranthum. Mean IZD ± SD (mm) Ampiclox® Extract (200 mg/mL) (1mg/mL) Staphylococcus aureus 33.70 ± 2.3 61.0 ± 5.3 Escherichia coli 33.70 ± 1.2 51.7 ± 5.8 Bacillus subtilis 5.76 ± 1.2 27.0 ± 3.5 IZD = inhibition zone diameter; SD = standard deviation. Test organisms


report in which methanolic extract of C. micranthum was assayed for in vitro antiviral activity against HSV-1 and HSV-2. In the test, the used methanolic extract after 7 days of storage produced high activity, which was not present in the freshly prepared extract. According to the authors, this phenomenon of increased antimicrobial activity after some days of storage of the extract might be due to the spontaneous transformations of inactive precursors in the extract to the active compounds by auto-oxidation. Since the antibacterial activity had been linked to tannins which are in abundance in the extract than any other metabolite, it might be reasonable to submit that the transformation of tannins to active forms was responsible for the observed steady increase in the antibacterial activity of the extract. This transformation might be due to the ability of tannins to evade the hydrolytic actions of certain enzymes like tannase (tannin acylhydrolase) which hydrolyse ester links of tannins. Hence, it becomes necessary to state that the extract of C. micranthum should be stored for at least 48 h (2 days) before the use for bioassay [10, 37].

the proanthocyanidins and tannins which were present in the extract. The antimicrobial activities of tannins are well documented [32-34]. Tannin is a polyphenol and has been well documented to have the microbicide activities against a huge number of bacteria, both gram positive and gram negative [10, 35]. The possible mechanism of their toxicity against microbes might be related to inhibition of hydrolytic enzymes (proteases and carbohydrates) or other interactions to inactivate microbial adhesions, cell envelope transport proteins and non-specific interactions with carbohydrates [10, 18]. The fact that extract produced antibacterial activity against both gram positive and gram negative bacteria is worthy of note because gram negative organisms are usually refractory to most antibiotics. 3.4 Effect of Storage Time of the Extract on Antibacterial Activity This test was carried out to determine if there would be significant (p < 0.05) change in antibacterial activity of the extract after storage for a predetermined time intervals of 1 h, 24 h, 48 h and 72 h, respectively. The obtained results are presented in Table 3. From Table 3, the result of the effect about storage time of the extract on the antibacterial activity of the plant on the test microorganisms showed an increase in antibacterial activity of the extract with increased duration of storage up to 48 h for S. aureus and B. subtilis, after which there was a declined inhibition during which most of these microbes have been cleared off. On the other hand, antibacterial activity of the extract against E. coli increased steadily throughout the duration of the study. Ferrea et al. [36] gave a similar Table 3

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3.5 Stability Testing of the Extract This study was carried out to determine the ability of the extract to resist microbial growth and degradation on storage alone and when some additives were incorporated. It was also done to determine whether the extract could be used for further studies after storage for about 1 month. On visual observation, a clear preparation was considered stable while a cloudy preparation was labeled unstable. The results were presented in Table 4.

Result of Effect of Storage time of extract on Antibacterial Activity. Mean IZD ± SD (mm)

Test organism

Extract (200 mg/mL)

Ampiclox® (1 mg/mL)

1h

24 h

48 h

72 h

S. aureus

28.3 ± 5.8

38.3 ± 5.8

41.4 ± 2.8

34.3 ± 3.1

51.7 ± 5.8

E. coli

30.3 ± 1.4

34.3 ± 0.0

39.4 ± 0.0

40.8 ± 1.7

38.0 ± 3.5

6.5 ± 1.0

7.2 ± 0.0

7.5 ± 1.2

7.0 ± 0.0

25.0 ± 2.0

B. subtilis

IZD = inhibition zone diameter; SD = standard deviation.

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Table 4 Result of stability Testing of the Extract alone and with some Additives. Preparations I II III IV V VI 1 2 3 4 5 + 6 + + 7 + + 8 + + 9 ++ + 10 ++ + 11 ++ ++ 12 +++ ++ 13 +++ ++ 14 +++ ++ 15 +++ + ++ 16 +++ + ++ 17 +++ + ++ 18 +++ + ++ 19 +++ + ++ 20 +++ + ++ 21 +++ + ++ 22 +++ + ++ 23 +++ + ++ 24 +++ + ++ 25 +++ + ++ 26 +++ + ++ 27 +++ + ++ 28 +++ + ++ 29 +++ + ++ 30 +++ + ++ Preparations: I = drug alone; II = drug + methyl paraben 0.3%; III = drug + propyl paraben 0.3%; IV = drug + sodium metabisulfite 0.01%; V = drug + methyl paraben 0.3% + propyl paraben 0.3%; VI = drug + methyl paraben 0.3% + propyl paraben 0.3% + sodium metabisulfite 0.01%; - = clear (no growth); + = slightly cloudy; ++ = cloudy; +++ = very cloudy. Day

The result of the Stability testing in Table 4 showed that the extract without any additive maintained its stability and resisted microbial growth for 4 days. Gradual growth and loss of stability commenced from the fifth day but became intense from the twelfth day, which signified total loss of stability. This might be due to the rapid hydrolytic actions of enzymes on the

metabolite contents of the extract which lead to the breakdown of their ester linkages [10, 18, 37]. This renders the extract to be susceptible to microbial growth and degradation. On the addition of methyl paraben which is an ester of p-hydroxybenzoic acid to the extract, the extract retained its stability throughout the duration of the study. This might be due to the activity of the methyl paraben as a preservative and the great permeability of cell membranes to the unionized molecules which are the active forms of the agent [38]. A combination of the extract and propyl paraben maintained the stability of the extract up to the fourteenth day. Loss of stability was observed from the fifteenth day which was slight for the remaining duration of study. This might be due to the tendency of the parabens to be very selective in action with increase in carbon chain of the esters. It might also be due to the increase in molecular weight of the paraben [39]. The addition of sodium metabisulphite to the extract maintained the stability of the extract for up to the fifth day. From the sixth day, there was gradual loss of stability, which increased from the eleventh day and maintained the same level to the last day of the experiment. This might be due to the concentration of the antioxidant which was insufficient to withstand the hydrolytic action of some enzymes which break down ester links of metabolites with antibacterial activity. In preparations V and VI, there was total stability due to the synergistic effect of combined preservatives [39].

4. Conclusions From the results of the various assays which carried out on the aqueous extract from the fresh leaves of Combretum micranthum, it could be concluded that the plant is rich in phenolic compounds, steroids and cardiac glycosides. The phenolic compounds had been demonstrated to be responsible for its antimicrobial activities which increase steadily with storage of the extract for at least 48 h (2 days). In addition, the stability of extract to degradation and microbial growth could be enhanced by the synergistic effects of methyl


paraben, propyl paraben and sodium metabisulphite.

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