1 Introduction

Marine algae produce a cocktail of metabolites with potential commercial value. Their medical and pharmaceutical applications have been investigated for a few decades (Bazes et al., 2009). Many compounds were discovered in the last years. The need for new drugs keeps this field open as many algal species are poorly screened. The ecological role of marine algal metabolites has somehow been overlooked. This new research field will provide valuable and novel insight into the marine ecosystem dynamics as well as a new approach to comprehending biodiversity. Furthermore, understanding interactions between compound production by algae and the environment, including anthropogenic or global climate changes, is a challenging target for the coming years.

Research on the active ingredients of metabolites has been more focused on macroalgae than on phytoplankton. However, phytoplankton could be a very promising material since it is the base of the marine food chain with quick adaptation to environmental changes, which undoubtedly has consequences on secondary metabolism (Cabrita et al., 2010).

Among all marine macroalgae, red algae are the main producers of bioactive compounds. Laurencia perforate is considered one of the most prolific genera (Faulkner, 2001 and Wright et al., 2003). A bioactive compound from Laurencia species is relatively high (Hill, 2007). Diterpenes, triterpenes, and C₁₅-acetogenins are the main secondary compounds of this genus (Faulkner, 1995) with antimicrobial, antifeedant, antihelmintic, and cytotoxic properties are generally associated (Davyt et al., 2001; Topcu et al., 2003 and Sun et al., 2005). Also, recent studies have shown promising antimalarial activity in Laurencia microcladia (Leon-Deniz et al., 2009).

One of the most important compounds of triterpens is β-amyrin. This compound is more potent than aspirin inhibiting collagen-induced platelet aggregation. In addition, β-amyrin includes skin care applications, anti-irritant, anti-inflammatory action as well as enhancement of the sun protection factor of organic sunscreens and emollient effect (Kweifio-Okai et al., 1995 and Ching et al., 2010). The present study aimed to evaluate the antibacterial activities as well as the chemical composition of β-amyrin isolated for the first time from L. microcladia.

2 Materials and methods

3 Results

In the present investigation, ethanolic extracts of 9 marine algal species were tested against 5 test organisms by agar diffusion method. The results of preliminary screening tests are summarized in Table 1, which revealed that, six algal species possess antibacterial activity. While the extracts of Codium fragile, Cladophora vagabunda, and Padina boryana did not show any activity during the experiment L. microcladia showed the largest clear zones against all tested Gram positive and Gram negative bacteria followed by Ulva linza and Cystoseira rayssiae. On the other hand, Acanthophora nayadiformis and Osmundea hybrid, were recorded moderate inhibitory activities, respectively. The extract of Sargassum angustifoium showed the lowest activities which appeared only against the two Gram positive bacteria. Regarding the susceptibility of the tested microorganisms, it was found that, the Gram positive bacteria were more sensitive to the algal extracts than the Gram negative (see Table 1).

As mentioned above, the red alga L. microcladia extract showed the highest antibacterial activity, and therefore the successive extractions of this extract using petroleum ether and chloroform in addition to the remained water extract were done. The experiment was conducted to detect the chemical analysis of the various components of the three extracts (Table 2), which proved that petroleum ether extract contained alkaloids, steroids, and/or triterpens while the chloroform extract contained only alkaloids. On the other hand, the aqueous extract did not contain any compound.

The successive ethanolic extract of L. microcladia was assayed for antibacterial properties (Table 3). The antibacterial activity depends on both algal species and efficiency of extraction of their active(s) principle(s). For example, petroleum ether extract was effective against all tested bacteria, while the chloroform extract was recorded to have inhibitory activity against Bacillus subtilis only. On the other hand, the remaining water extract had no activity.

3.1

3.1 Chemical analysis

Chemical analysis of the petroleum ether extract of the alga Laurencia confirmed the extract to contain triterpens. The antibacterial test confirmed high effectiveness of this extract to discourage the growth of bacteria. These results lead to complete separation, purification, and identification of active substances contained in the petroleum ether extract.

The isolated compound was identified according to the melting point (191–194 °C m.p.), infrared spectrum (IR), nuclear magnetic resonance (¹H NMR) of the proton and mass spectrometry (MS). The infrared spectrum recorded in the presence of potassium bromide was as follows: IR (KBr): √max 3292 and 1035 cm⁻¹ (OH group) and 2945, 2850, 1460 and 1385 cm⁻¹ (aliphatic methylene and methyl group). ¹H NMR methyl single at 0.98, 0.79, 0.92, 0.96, 1.12, 0.82, and 0.86 and olefinic proton resonating at δ 5.17 (1H, t, J = 4 Hz, H-12). MS: m/z (rel int %) 218 (100), 203, 207, and 189 (pentacyclic triterpene amyrin) as described by Krishnaswamy (1999). The molecular formula indicates that it is a pentacyclic triterpene with molecular formula C₃₀H₅₀O, and this is supported by the characteristic play of colors given by the compound on treatment with the Liebermann–Burchard reagent (acetic anhydride–sulfuric acid). This compound can be identified as β-amyrin and the chemical structure is given in Fig. 1.

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Figure 1

Molecular structure of β-amyrin.

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The minimum inhibitory concentration (MIC) of the isolated compound was detected (Table 4). The data reported that the MIC against Staphylococcus aureus and Salmonella typhi was 2.5 mg mL⁻¹.

Table 4 Minimum inhibition concentration of the isolated β-amyrin.

Compound concentration mg mL−1	Diameter of inhibition zone (mm)
	Staphylococcus aureus	Salmonella typhi
10	26	20
5	20	16
2.5	14	11
0.125	−ve	−ve

−ve, no inhibition zone.

4 Discussion

The present data revealed a high antibacterial activity from the ethanolic extract of the red alga L. microcladia (Table 1). It is noteworthy to mention that this alga exhibited a broad spectrum of antibacterial activity (Abdel-Raouf et al., 2008). It was confirmed by the successive extracts by petroleum ether, chloroform, and water (Table 3) which revealed the affectivity of petroleum ether in the extracts of the bioactive materials. The preliminary phycochemical screening of the successive extracts prepared from L. microcladia (Table 2) indicated the presence of alkaloids, steroids, and/or triterpens. Additionally, the chemical analysis of the petroleum ether extract indicated that the bioactive ingredient was β-amyrin. So the broad spectrum of antibacterial activity recorded by L. microcladia and their beneficial effects could be attributed to the presence of β-amyrin which has inhibitor properties retarding the growth of these bacteria and antagonizing the infection mechanisms of these microbes. Our finding agreed with Kurata et al. (1998) and Hill (2007), who reported that, the bioactive compounds from genus L. microcladia were relatively high with diterpens, triterpenes, and C₁₅-acetogenins. It was reported that these compounds exhibited antimicrobial, antifeedant, antihelmintic, and cytotoxic properties (Sun et al., 2005 and Cabrita et al., 2010).

In this study, Codium fragile, Cladophora vagabunda, and Padina boryana did not show any activity during the experiment. The variation in the antibacterial activity between the studied algal species may be due to the method of extraction, solvent used and season at which samples were collected (Febles et al., 1995; Lima-Filho et al., 2002; Tuney et al., 2006).

In the present study, it was observed that Gram positive bacteria were more sensitive to the algal extracts than the Gram negative. In this respect, Ozdemir et al. (2006), Salvador et al. (2007), Abdel-Raouf and Ibraheem (2008) and Ibraheem et al. (2012) reported that Gram-positive bacteria are more effectively controlled by the studied algal extracts compared to Gram negative bacteria. Similar observations also were recorded by Taskin et al. (2001, 2007), indicating that more sensitivity of Gram positive bacteria towards the algal extract was due to the differences in their cell wall structure and their composition. Gram negative cell walls are more complex than Gram positive cell walls, both structurally and chemically. Structurally, a Gram negative cell wall contains two layers external to the cytoplasmic membrane. Immediately external to the cytoplasmic membrane is a thin peptidoglycan layer, which accounts for only 5% to 10% of the Gram negative cell wall by weight. There are no teichoic or lipoteichoic acids in the Gram negative cell wall. External to the peptidoglycan layer is the outer membrane, which is unique to Gram negative bacteria. This outer membrane acts as a barrier to many environmental substances including antibiotics (Tortora et al., 2001).

At determination of LD₅₀ of the isolated compound, it was found that, this bioactive compound failed to kill rats within 72 h in doses up to 395 mg kg⁻¹. This means that, the studied extract is considered to be safe for human use (LD₅₀ is very low). This agrees with the findings of Galvin and Mikhail (1976) who mentioned that the substances possessing safety test up to 2000 mg kg⁻¹ are considered non toxic.

5 Conclusions

Finally it can be concluded that, the extracts of L. microcladia used in the present investigation showed better antibacterial activity against pathogens used. The potential source of bioactive compound β-amyrin should be investigated with further studies.