Antibacterial, Antifungal, and Cytotoxic Activities of 11 Solanaceae Plants from Colombian Biodiversity

Abstract

The hexane, dichloromethane, and methanol extracts of 11 Solanaceae plants collected in Regional Natural Park Ucumarí (RNPU) Colombia were evaluated for their antibacterial and antifungal activities through the agar-well diffusion method and for cytotoxic activity by the brine shrimp lethality assay. In general, the methanol plant extracts were more bioactive in the three different tests performed in this work. Under conditions and concentrations of the plant extracts evaluated in this research, the cytotoxic activities against brine shrimp Artemia salina. (Leach) larvae were the strongest compared with the antibacterial or the antifungal activities. The strongest cytotoxic activities were observed with the methanol extracts from Browallia speciosa. Hook (LC₅₀ 0.01 mg/ml) and Deprea glabra. (Standl.) A.T. Hunziker (LC₅₀ 0.01 mg/ml). The three extracts from Solanum deflexiflorum. Bitter (MIC 0.31 mg/ml) and the dichloromethane extract from Solanum leucocarpun. Dunal (MIC 0.31 mg/ml) were more bioactive against the Gram-positive bacteria Bacillus subtilis.. The methanol extract from Lycianthes synanthera. (Send.) Bitter exhibited good antimycotic activities against Candida albicans. and Fusarium solani. both with a MIC of 0.62 mg/ml.

Keywords:

• Agar-well diffusion method
• antimicrobial screening
• Artemia salina
• bioassay
• bioprospecting
• Colombian flora
• natural products

Introduction

The Solanaceae family is widely distributed in hot and temperate zones around the world and includes the large genus Solanum. (Griffin & Lin, 2000) that is rich in steroidal glycoalkaloids, which is important from the ecological and the commercial point of view (Fukuhara et al., 2004).

From the ecological aspect, steroidal glycoalkaloids are considered defense allelochemicals against a great number of pathogens and predators (Eltayeb et al., 1997; Weissenberg et al., 1998). From the commercial view, they are a source of raw materials for the semisynthesis of pharmaceutical and contraceptive steroids (Eltayeb et al., 1997; Okršlar et al., 2002). In addition, some glycoalkaloids display a wide range of biological activities such as antitumor, anticancer, and anti-herpes (Ikeda et al., 2003), as well as teratogenic, embryotoxic, antifungal, and molluscicidal (Kim et al., 1996).

Due to the remarkable variety and complexity of compounds isolated from plants and to the fact that many of the current drugs used to treat different types of diseases are from plant sources, there is a great potential to discover new substances to be used in the treatment of bacterial or fungal infections (Kinghorn et al., 1999; Koné et al., 2004). In addition, there is a constant need to discover new antimicrobials from natural sources, due to the notable ability of microbes to develop resistance against recently available antibiotics (Saves & Masson, 1998).

The extensive use of the Solanaceae plants in folk medicine, the clinical effect shown by the steroidal alkaloids from the Solanaceae family, the accelerated forest destruction, and the great Colombian biodiversity flora prompted us to perform some biological screenings of plants belonging to the Solanaceae family from the Regional Natural Park Ucumarí (RNPU) against seven microorganisms and through the brine shrimp lethality assay.

Materials and Methods

Plant material

Eleven plants belonging to the Solanaceae family were collected in Regional Natural Park Ucumarí (RNPU) in October 2001 and authenticated by Dr. F.J. Roldán. Voucher specimens for each plant collected were deposited at the Universidad de Antioquia Herbarium (Medellín, Colombia), (Table 1).

Table 1 Minimum inhibitory concentration (MIC, mg/ml) and lethal dose 50 (LD₅₀, mg/ml) of crude plant extracts of Solanaceae family collected at Regional Natural Park Ucumarí (RNPU)

		Bacteria (MIC, mg/ml)				Fungi (MIC, mg/ml)
Species (Voucher no.)	Extract	S. aureus.	B. subtilis.	E. coli.	P. aeruginosa.	C. albicans.	A. fumigatus.	F. solani.	Artemia salina. (LD50, mg/ml)
Solanum ovalifolium. Dunal (FJR3714)	H	–	–	–	–	–	–	–	0.50
	DC	–	–	–	–	–	–	–	1.00
	Mt	–	–	–	–	0.62	–	–	0.76
Solanum leucocarpun. Dunal (FJR3717)	H	–	1.25	–	–	–	–	–	0.44
	DC	–	0.31	–	–	–	–	–	1.00
	Mt	–	0.62	–	–	–	–	–	0.52
Solanum deflexiflorum. Bitter (FJR3718)	H	–	0.31	–	–	–	–	–	0.68
	DC	–	0.31	–	–	0.62	5.00	–	1.00
	Mt	5.00	0.31	–	–	–	5.00	–	0.43
Lycianthes synanthera. (Send.) Bitter (FJR3719)	H	–	–	–	–	–	–	–	1.00
	DC	5.00	1.25	–	–	1.25	–	0.62	0.52
	Mt	5.00	1.25	–	–	0.62	–	0.62	0.48
Deprea glabra. (Standl.) A.T. Hunziker (FJR3722)	H	–	–	–	–	–	–	–	0.40
	DC	–	–	–	–	–	–	–	0.66
	Mt	–	–	–	–	–	5.00	–	0.01
Browallia speciosa. Hook (FJR3732)	H	–	–	–	–	–	–	–	0.52
	DC	–	5.00	–	–	–	2.50	–	0.26
	Mt	–	2.50	–	–	2.50	2.50	–	0.01
Solanum lepidotum. Dunal (FJR3728)	H	–	–	–	–	–	–	–	1.00
	DC	–	–	–	–	–	–	–	0.46
	Mt	–	–	–	–	2.50	–	2.50	0.20
Solanum aphyodendron. S. Knapp (FJR3729)	H	–	–	–	–	–	–	–	0.58
	DC	–	–	–	–	–	–	–	1.00
	Mt	–	–	–	–	2.50	–	–	0.14
Lycianthes sp.. (FJR3735)	H	–	–	–	–	–	–	–	0.62
	DC	–	2.50	–	–	–	–	1.25	1.00
	Mt	–	1.25	–	–	–	–	1.25	0.28
Solanum sycophanta. Dunal (FJR3737)	H	–	–	–	–	–	–	–	1.00
	DC	–	–	–	–	2.50	–	–	1.00
	Mt	–	–	–	–	1.25	–	–	0.40
Solanum stellatiglandulosum. Bitter (FJR3717)	H	–	–	–	–	–	–	–	1.00
	DC	–	–	–	–	–	–	–	1.00
	Mt	–	2.50	–	–	2.50	5.00	–	0.52
Positive control	Cefotaxime	0.25	5.00	0.50	0.50	NE	NE	NE	NE
	Ketokonazole	NE	NE	NE	NE	0.25	0.25	0.25	NE
	Gallic acid	NE	NE	NE	NE	NE	NE	NE	0.1

H, hexane; DC, dichloromethane; Mt, methanol; –, no growth inhibition at the concentrations tested; NE, not evaluated.

Extract preparation

The aerial plant materials were oven-dried at 50°C then ground and extracted by lixiviation at room temperature three-times with each of the following solvents: hexane, dichloromethane, and methanol. Then, the different extracts were concentrated at reduced pressure to dryness and stored at − 10°C until assayed.

Microorganisms and Artemia salina. (Leach)

For antibacterial screening; tests were performed with Gram-positive Bacillus subtilis. (ATCC 21556), Staphylococcus aureus. (ATCC 6538), and the Gram-negative Pseudomonas aeruginosa. (ATCC 27853) and Escherichia coli. (ATCC 9637). For antimycotic activity, tests were performed with Candida albicans. (ATCC 18804), Aspergillus fumigatus. (ATCC 1022), and Fusarium solani. (ATCC 11712). The cytotoxic assays were performed with Artemia salina. (Leach) larvae (Brine Shrimp Eggs, San Francisco, CA, USA).

Antimicrobial activity

The antimicrobial activities of the crude hexane, dichloromethane, and methanol extracts were evaluated through the agar-well diffusion assay (Ríos et al., 1988). The procedures for the antimycotic and antibacterial assays were described by Niño et al. (2003) and Mosquera et al. (2004), respectively. The extracts were resuspended in 95% ethanol and tested at five different concentrations (5.00, 2.50, 1.25, 0.62, and 0.31 mg/ml). For the antibacterial and antifungal assays, as positive control, cefotaxime and ketokonazole were used, respectively.

The results for the antibacterial and antimycotic assays were recorded for each plant extract by measuring (mm) the zones of growth inhibition surrounding each well. The tests were carried out in triplicate, and the mean of the inhibition zones was calculated. Then, the minimum inhibitory concentration (MIC) was obtained.

Cytotoxic assay

The cytotoxic in vivo. brine shrimp lethality tests were carried out by using brine shrimp Artemia salina. (Leach) larvae, according to the methodology described by McLaughlin et al. (1994). Each plant extract was tested at 1.00, 0.10, and 0.01 mg/ml and also evaluated by triplicate. The methanol plant extracts were dissolved in water and those from hexane and dichloromethane were dissolved in the same solvents following evaporation for 16 h. After that, 10 ml of artificial sea water were added to each vial. As positive control, gallic acid was used. In all cases, as a negative control, each solvent to dissolve the original extract was used.

Mortality percentage (% M) for each concentration was calculated by applying the Abbot formula. Lethal concentration 50 (LC₅₀; in mg/ml) was determined for each plant extract by interpolation in the graph from the mortality percentage versus the concentration in mg/ml and through a linear regression analysis with the program Microsoft Excel. The cytotoxic activity was considered weak when the LC₅₀ was between 0.50 and 1.00 mg/ml, moderate when the LC₅₀ was between 0.10 and 0.50 mg/ml, and designated as strong when the LC₅₀ ranged from 0 to 0.10 mg/ml (Padmaja et al., 2002).

Phytochemical screening

These tests were performed on different extracts obtained from the Solanaceae plants collected and following the procedures described by Harborne (1980) and Domínguez (1985).

Results and Discussion

Table 1 summarizes the antimicrobial and cytotoxic activities displayed by the Solanaceae plant extracts that were evaluated in this research. Methanol extracts were the most active (100%) in microbiological tests than those from dichloromethane (54.54%) or hexane (18.18%); moreover, the methanol extracts were also the most cytotoxic. This could be due to the methanol polarity that is able to extract many different polar constituents of the plant having antimicrobial activities.

Solanum deflexiflorum. Bitter and Lycianthes synanthera. were the only plant species that inhibited four of the seven microorganisms evaluated in this research. In addition, S.. deflexiflorum. and Solanum leucocarpun. Dunal were the only plant species that showed inhibition with their hexane extracts.

Antibacterial

From 33 plant extracts tested, only 13 (39.39%) displayed activity against Gram-positive bacteria, with B. subtilis. being the most susceptible with 13 (39.39%) extracts showing inhibition, followed by S. aureus. with 3 (9.09%) extracts. These findings are in agreement with the fruits methanol extracts of Solanum torvum. that exhibited activity against both B. subtilis. and S. aureus., with a MIC equal to 0.31 mg/ml, supporting some of the antimicrobial activities attributed to this plant (Chah et al., 2000; Wiart et al., 2004). In addition, these results correlate with those from selected Peruvian medicinal plants where Lochroma umbellatum. (Solanaceae) displayed great antimicrobial activity against B. subtilis. and S. aureus. (Rojas et al., 2003).

The lowest MIC (0.31 mg/ml) values of B. subtilis. were obtained with the three different extracts from S. deflexiflorum. and the dichloromethane extract from S. leucocarpun.. According to phytochemical screening (data not shown), the dichloromethane and methanol extracts from both species showed evidence of alkaloids, sterols, triterpenes, and steroids.

Not one of the plant extracts was active against the Gram-negative E. coli. and P. aeruginosa.. These results correlate with those found in root and stem extracts of Datura stramonium. where P. aeruginosa. was observed to be completely resistant to the extract of this Solanaceae (Obi et al., 2002) as well as the methanol extract of Capsicum annum. (Solanaceae) (Shahidi et al., 2004). In addition, the same Gram-negative bacteria presented small inhibition zones with the methanol extracts of Solanum macrocarpum. leaves (Ajaiyeola, 1999) and with the ethanol extracts of Withania somnifera. leaves, which gave weak activity against P. aeruginosa. and E. coli. (Awadh et al., 2001).

Antimycotic

The most susceptible fungus was C. albicans. that showed activity with 10 extracts (30.30%). The lowest MIC values were observed with the methanol extracts from Solanum ovalifolium. Dunal and L. synanthera. (0.62 mg/ml),respectively, and the dichloromethane extract from S. deflexiflorum. (0.62 mg/ml) (Table 1). The phytochemical screening of the methanol extracts from the former two plants showed the presence of alkaloids, sapogenins, and tannins (data not shown). These findings are in agreement with the significant antimycotic activity against C. albicans. and the filamentous fungus Mycrosporum gypseum. from the crude ethanol extract of Cestrum auriculatum. (Solanaceae) (Rojas et al., 2003). In addition, the dichloromethane and methanol extract from L. synanthera. gave a low MIC (0.62 mg/ml) against F. solani.. These results correlate with the fungitoxic activity given by D. stramonium. extracts against five pathogenic fungi (Shivpuri et al., 1997).

Cytotoxicity

The strongest cytotoxic activities were showed by the methanol extracts of Browallia speciosa. Hook and Deprea glabra. (Standl.) A.T. Honziker, both with LC₅₀ equal to 0.01 mg/ml (Table 1); not one of these two extracts showed proof of the secondary metabolites under phytochemical conditions assayed in this research (data not shown).

The dichloromethane extracts from B. speciosa. and Solanum lepidotum. Dunal as well as the hexane extracts from D. glabra. and S. leucocarpun. all gave moderate cytotoxicity against Artemia salina.. The phytochemical screening of dichloromethane extracts of B. speciosa. showed evidence of saponins while that of S. lepidotum. displayed the presence of sterols, steroids, saponins, and triterpenes. These findings are consonant with those found by Moreno-Murillo et al. (2001), where the ethanol extracts and chromatographic fractions of Solanaceae Nicandra physaloides., Salpichora diffusa., and Salpichora tristis. var tristies displayed high toxicity in the brine shrimp test. In addition, Solanum luteum. Mill. was toxic (0.055 mg/ml) in the brine shrimp bioassay (Alkofahi et al., 1996), and diurnoside, a steroidal saponin from Cestrum diurnum. (Solanaceae), exhibited toxicity in the brine shrimp assay (Ahmad et al., 1994).

Conclusions

The strongest antimicrobial activity of all the species of Solanaceae examined was observed with Solanum deflexiflorum. followed by Lycianthes synanthera. and Solanum leucocarpun.. It is important to mention that the three different extracts from S. deflexiflorum. were the only plant extracts from the 33 examined that showed strong activity particularly against B. subtilis..

The Solanaceae plant species tested in fact possess antibacterial, antifungal, and cytotoxic activities against the organisms examined. These findings are the base for further studies to isolate (guided by biological assays) and elucidate, the structure of the bioactive compounds assessed from these plants.

Our results are a contribution to a better understanding of the Colombian biodiversity, as some of them may become an important source of antimicrobial or cytotoxic agents.

Acknowledgments

The authors acknowledge Universidad Tecnológica de Pereira for its financial support to this research and the CARDER for granting the permissions to access plant collection.