Bioactivities of Ethanolic Extract and its Fractions of Cistus laurifolius L. (Cistaceae) and Salvia wiedemannii Boiss. (Lamiaceae) Species.

BACKGROUND: Cistus laurifolius L. (Cistaceae) and Salvia wiedemannii Boiss. (Lamiaceae) have been used for treatment of some illnesses in Turkish folk medicine. In the present study, the ethanolic extract and its fractions obtained using re-extraction by hexane (Hx), chloroform (CHCl3), butanol, and remaining-water (r-H2O) of C. laurifolius were screened for their in vitro bioactivities.
MATERIALS AND METHODS: Activities were determined against both standard and the isolated strains of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, as well as yeasts such as Candida albicans and Candida parapsilosis by microdilution method. Also, antiviral activity of C. laurifolius and S. wiedemannii extracts were tested on herpes simplex virus-1 (HSV-1) and parainfluenza-3 (PI-3) using Madin-Darby bovine kidney and vero cell lines.
RESULTS: Tested extracts of C. laurifolius (minimum inhibitory concentration 32 μg/mL) exerted a strong antimicrobial activity against Gram-negative bacteria of E. coli, P. mirabilis, K. pneumoniae, and A. baumannii.
CONCLUSION: The Hx extract of C. laurifolius (cytopathogenic effect of 32-8 μg/mL) had antiviral activity on PI-3. Also, the r-H2O, CHCl3, and ethanol extracts (16-<0.25 μg/mL) of S. wiedemannii had significant antiviral activity on HSV-1, same as control.
SUMMARY: The objective of this study was to evaluate the bioactivity of plant extracts used in folk medicineEthanolic extract and its fractions obtained using re-extraction by hexane (Hx), chloroform (CHCl3), butanol, and remaining-water (r-H2O) of Cistus laurifolius L. (Cistaceae) and Salvia wiedemannii Boiss. (Lamiaceae) were screened against both standard and the isolated strains of E. coli, P. aeruginosa, P. mirabilis, K. pneumoniae, A. baumannii, S. aureus, E. faecalis, C. albicans and C. parapsilosis by microdilution methodAntiviral activity were tested on HSV-1 and PI-3 using MDBK and Vero cell linesExtracts of C. laurifolius exerted a strong antimicrobial activity against E. coli, P. mirabilis, K. pneumoniae, and A. baumannii(MIC; 32 μg/mL)The Hx extract of C. laurifolius had antiviral activity on PI-3 (CPE; 32-8 μg/mL). Also, the r-H2O, CHCl3, and ethanol extracts (16-<0.25 μg/mL) of S. wiedemannii had significant antiviral activity.

INTRODUCTION

Resistances to current antimicrobiological agents have been increasing. Therefore, there is a need for developing new antimicrobial drugs. Plants can be a source of these new medications given the fact that plants have been used as active substance of numerous antibiotics by industry[12] and as “anti-infection drugs” by people in folk medicine.

Five Cistus species belonging to Cistaceae family grow naturally in Turkey. The genus Salvia widely distributed in Turkey is represented by 94 taxa belonging to 89 species, with a 50% ratio of endemism.[3] Cistus and Salvia species have been used against high fever, rheumatic pain, peptic ulcer, stomachache, urinary inflammations, catarrh, cold, wounds, stomachache, flatulence, constipation, rheumatic pain, wards, sunstroke, and hemorrhage in Turkish folk medicine.[45678910] It has been shown that Cistus and Salvia species have some activities, including antimicrobial[11121314] antioxidant,[11141516] anti-inflammatory,[171819] analgesic,[182021] and antiviral[1422232425] activities.

In our previous studies, we demonstrated in vitro inhibitory effects of Cistus laurifolius leaves on interleukin-1 alpha and beta (IL-1α, and IL-1β) and tumor necrosis factor (TNF) biosynthesis. The extract and fractions obtained from the leaves were found to be ineffective on TNF inhibition, but effective on IL-1 inhibition. The methanolic extract and the hexane (Hx) and chloroform (CHCl3) fractions were found to have remarkable IL-1α inhibitory effects in high concentrations.[17] Three active compounds were isolated from the CHCl3 extract of C. laurifolius. Of these three compounds, quercetin 3-methyl ether exhibited the highest inhibitory effect on H. pylori at the concentration of 3.9 μg/mL.[13] Likewise, analgesic activity of the same extract was also demonstrated. C. laurifolius CHCl3 extract and its precipitated fraction exhibited central analgesic effect in mice.[20] These studies support folkloric utilization of the plant. In addition to these confirm activities, we aimed to evaluate bioactivities of these plants.

In this study, antibacterial and antifungal activities of the ethanol (EtOH), Hx, CHCl3, buthanol (BuOH), and remaining-water (r-H2O) extracts of C. laurifolius were assessed against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and their isolates as well as yeasts namely; Candida albicans and Candida parapsilosis. Also, we evaluated the efficacy of C. laurifolius and Salvia wiedemannii extracts (EtOH, Hx, CHCl3, BuOH, and r-H2O) on Herpes simplex virus-1 (HSV-1) and Parainfluenza-3 (PI-3) viruses. The effects of these extracts were compared to reference drugs (i.e. ampicillin, ofloxacin, levofloxacin, ketoconazole, fluconazole, acyclovir, and oseltamivir).

MATERIALS AND METHODS

Plant material

The leaves of C. laurifolius and the aerial parts of S. wiedemannii were collected in the vicinity of Kurtboğazı (Ankara-May 2005) and Yunak (Ankara-May 2003) in Turkey, respectively. Voucher specimens were identified by a comparison with authentic specimens that had already been identified by Prof. Dr. Ekrem Sezik. Authenticated voucher specimens (C. laurifolius-GUEF No: 97B002, S. wiedemannii-GUEF No: 2379) were stored in the herbarium of Faculty of Pharmacy at Gazi University, Ankara, Turkey.

Preparation of the extract and its fractions

The air-dried and powdered plant (C. laurifolius; 510 g, S. wiedemannii; 360 g) was macerated in 95% EtOH (2200 mL EtOH) for 6 h in water bath adjusted to 40°C. Then, the macerate was filtrated, and this procedure was repeated three times. The combined extracts were evaporated to dryness in vacuo using a rotary evaporator (C. laurifolius EtOH extract 32.35%, S. wiedemannii EtOH extract 31.38%). The EtOH extract was redissolved in 90% MeOH/H2O (1000 mL) and re-extracted with portions of n-Hx (3 × 300 mL) for 6 h in water bath adjusted to 40°C. The combined Hx fraction was evaporated to dryness to obtain a sticky residue (the Hx fraction for C. laurifolius and S. wiedemannii; 4.5% and 3.61%, respectively). Then, the MeOH/H2O extract was concentrated under reduce pressure in a rotary evaporator and dissolved in H2O (150 mL). The water extract was re-extracted with portions of CHCl3 (3 × 400 mL) for 6 h in water bath adjusted to 40°C. The combined CHCl3 fraction was evaporated in a rotary evaporator (CHCl3 fraction for C. laurifolius and S. wiedemannii; 10.98%, and 10.27%, respectively). The aqueous extract was then re-extracted with BuOH saturated with distilled H2O for 6 h in water bath adjusted to 40°C and evaporated to dryness (BuOH fraction for C. laurifolius and S. wiedemannii; 6.27%, and 7.77%, respectively). The remaining aqueous part (r-H2O) was lyophilized (r-H2O fraction for C. laurifolius and S. wiedemannii; 9.6% and 8.61%, respectively).

Bioactivities assay

The ethanolic extract, and Hx, CHCl3, BuOH, and r-H2O fractions dissolved in dimethylsulfoxide (DMSO, 80%) and EtOH (20%) at a final concentration of 512 μg/mL. The extracts were sterilized by filtration using 0.22 μm millipore used as the stock solutions. Reference pharmaceutical agents were purchased from Sigma Chemical Co., and dissolved in phosphate buffer solution (ampicillin, pH: 8.0; 0.1 mol/mL), DMSO (ketoconazole) or in water (ofloxacin, levofloxacin, and fluconazole).[2627] As the standards, Gram-negative strains of E. coli ATCC 35218, P. aeruginosa ATCC 10145, P. mirabilis ATCC 7002, K. pneumoniae RSKK 574, A. baumannii RSKK 02026, and as Gram-positive strains of S. aureus ATCC 25923, and E. faecalis ATCC 29212, and its isolates were used for the determination of antibacterial activity. C. albicans ATCC 10231 and C. parapsilosis ATCC 22019 were used for determination of antifungal activity. Mueller Hinton Broth (MHB; Difco) and Mueller Hinton Agar (MHA; Oxoid) were applied for growing and diluting of the bacteria suspensions. The synthetic medium RPMI-1640 with L-glutamine was buffered to pH: 7 with 3-(N-morpholino)-propansulfonic acid and culture suspensions were prepared as described previously.[2627] The microdilution method was employed for antibacterial and antifungal activity tests as described previous study.[2829]

Vero cell line and Madin-Darby bovine kidney cell cultures were grown in Eagle's Minimal Essential Medium (Seromed; Biochromin) enriched with 10% fetal calf serum (Biochrom, 100 mg/mL of streptomycin; 100 IU/mL of penicillin) in a humidified atmosphere of 5% carbon dioxide at 37°C as described previously. In order to determine the antiviral activity of the extracts, HSV-1, and PI- 3 were used. Maximum cytopathogenic effect (max CPE) concentrations as the indicator of antiviral activities of the extracts were determined with maximum nontoxic concentrations (MNTCs) as described previously.[2829]

RESULTS

The results of comparative antibacterial and antifungal activities of the extracts of C. laurifolius, which assayed in vitro antibacterial and antifungal activity against standard strains of E. coli P. aeruginosa, P. mirabilis, K. pneumoniae, A. baumannii, S. aureus, E. faecalis and their drug-resistant strains as well as yeasts C. albicans, and C. parapsilosis are presented in Table 1.

Table 1

Antimicrobial activity of the C. laurifolius extracts and references expressed as minimum inhibitory concentrations (MICs; μg mL−1)

As shown in Table 1, all of the extracts of C. laurifolius showed the same activity against standard Gram-negative bacteria (E. coli, P. mirabilis, K. pneumoniae, and A. baumannii) with minimum inhibitory concentrations (MICs) value of 32 µg/mL. The efficacies of the extracts were lower against resistant isolates (ESβLs enzyme positive; E. coli, trimethoprim-sulfamethoxazole, tazobactam-resistant P. aeruginosa, trimethoprim-sulfamethoxazole, cefriaxon-resistant P. mirabilis/K. pneumonia, and trimethoprim-sulfamethoxazole-resistant A. baumannii). MICs for all microbes were two times higher for all extracts. The extracts of C. laurifolius showed some degree of antibacterial activity against standard and isolated strains of P. aeruginosa at MIC values of 64, 128 µg/mL, respectively. Interestingly, the extracts were found to have good activity against isolated Gram-negative strains of E. coli, which showed a close effect to those of the references; ampicillin (MIC; 64 µg/mL).

As for Gram-positive bacteria, MIC values of 64 μg/mL and 128 μg/mL were determined for standard (S. aureus ATCC25923, and E. faecalis ATCC29212) and drug-resistant strains (methicillin-resistant S. aureus, and cephalosporin-resistant E. faecalis), respectively.

All extracts had similar MICs values (8 µg/mL) on C. albicans and C. parapsilosis compared to the references drugs employed (ketoconazole and fluconazole).

The results of antiviral activities of the extracts of C. laurifolius, and S. wiedemannii are presented in Table 2. As shown in Table 2, the H2O, CHCl3, and EtOH extracts (16–<0.25 μg/mL) of S. wiedemannii had significant antiviral activity on HSV-1 with a MNTC of 16 µg/mL, similar to that of acyclovir. The BuOH extract of S. wiedemannii had remarkable inhibitory activity on HSV-1 (32–<0.25 μg/mL). On the other hand, the BuOH and Hx extracts of C. laurifolius had less antiviral activity (32–16 µg/mL) on HSV-1 with the MNTC of 32 µg/mL. In particular, the Hx extract of C. laurifolius (CPE of 32–8 µg/mL) had significant antiviral activity on PI-3 with a MNTC of 32 µg/mL, similar to that of oseltamivir (CPE of 32–<0.25 µg/mL). Regarding PI-3, the EtOH, BuOH extracts of C. laurifolius, and the BuOH extract of S. wiedemannii showed the same inhibitory activity (CPE of 64–16 µg/mL).

Table 2

Antiviral activity together with CPEs of the extract of the C. laurifolius and S. wiedemannii and references as MICs (μg mL−1) values

DISCUSSION

In a previous study, in vitro bioactivities of the aqueous extracts of C. ladanifer and C. populifolius leaves were studied.[11] Their cytotoxicity on human cancer cells was determined. C. ladanifer showed remarkable antibacterial activity on S. aureus, whereas C. populifolius was effective against E. coli with MICs values of 154 and 123 µg/mL, respectively. Also, cytotoxic activity of both extracts was observed against breast cancer cells.

The water, methanol, CHCl3, ethyl acetate, and butanol extracts, obtained from leaves and the fruits of the five Cistus species (C. creticus L., C. laurifolius L., C. monspeliensis L., C. parviflorus Lam., and C. salviifolius L.), were investigated against the following microorganisms: S. aureus, Streptococcus faecalis, Bacillus subtilis, B. cereus, P. aeruginosa, E. coli, and C. albicans with disc diffusion method.[12] All extracts of C. laurifolius showed activity against S. aureus, whose inhibition zones varied between 8 mm and 14 mm. Additionally, methanol, CHCl3 extract, ethyl acetate extract, n-butanol extract, and water extracts exerted activity against B. subtilis (between 8 mm and 11 mm) and B. cereus (between 9 mm and13 mm). Only the butanol extract showed activity against S. faecalis (8 mm) and E. coli (11 mm). Cefazolin (S. aureus; 23.21 mm, B. subtilis; 23 mm, B. cereus; 20 mm) and ciprofloxacin (S. faecalis; 22 mm, E. coli; 27 mm) used as the references. None of the extracts of C. laurifolius were active against P. aeruginosa and C. albicans. As a matter of fact, no comparison could be made between the results of our and the aforementioned study,[12] depending on difference on the methods used in both studies. In our study, all extracts exhibited antimicrobial activity.

C. incanus extract which was rich in polyphenolics exerted antiviral activity against influenza A virus (H7N7) in vitro and in vivo.[22] Relevantly, antiviral activity of Salvia species has been evaluated. Ogutcu et al. studied the methanol, Hx, and dichloromethane extracts of S. limbata and S. sclarea. Anti-influenza drug rimantadine and (E)-5-(2-bromovinyl)-2‘-deoxyuridine hydrochloride were used as the references. The dichloromethane extract of S. sclarea and the methanol extract of S. limbata showed high anti-influenza virus activity while the methanol extract of S. sclarea had a limited antiherpetic activity.[14] The inhibitory activity of the aqueous extract of S. officinalis on HSV-1 and HSV-2 was tested using RC-37 cells (African green monkey kidney cells). In the study, acyclovir was the positive control. The aqueous extracts of S. officinalis had significant antiviral activity on HSV-1 (0.777 g/mL) and HSV-2 (1.359 μg/mL).[23] In another study, antiviral activities of seven extracts of S. miltiorrhiza were evaluated.[24] The ethyl acetate and water extracts of the plant inhibited viral RNA synthesis; however, the other extracts did not have any protective activity. Fifty percentage inhibitory concentrations of the ethyl acetate and water extracts on Enterovirus 71 were 0.742 ± 0.042 mg/mL and 0.585 ± 0.018 mg/mL, respectively. Likewise, another study showed antiviral activity (anti-human deficiency virus) with no cytoxicity of two compounds (lithospermic acid and lithospermic acid B) isolated from the roots of S. miltiorrhiza.[25] The CC100 doses for lithospermic and lithospermic acid B were >297 μM and >223 μM, respectively.

Recently, resistance to conventional antiviral drugs is on rise. Therefore, there is a strong need for novel effective substances synthesized or isolated from plants. This study shows for the first time that C. laurifolius and S. wiedemannii are very promising plants regarding their antiviral and cytotoxic effects. However, further studies are necessary on the isolation and mechanism of the active component (s) of C. laurifolius and S. wiedemannii.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Dr. Berrin Özçelik

ABOUT AUTHOR

Prof. Dr. Berrin Özçelik, Departments of Pharmaceutical Microbiology, Gazi University, 06330 Ankara, Turkey