Introduction

Flowers and plants are the most telling symbol of power and greatness of the creation. The value of this gift from God, not only in human and animal food supply But the cure and relieve pain in many human person can be found in plants. Study and research on drugs that are vegetable or animal origin is done in the field of Pharmacognosy and Pharmacology .This process of evolution today is one of the specific fields of pharmacy education in several main branches. Although the vast majority of drugs are chemical but it’s estimated that at least a third of all pharmaceutical products are from herbal sources or after extraction of plants are modified.Biotechnology tries to produce new group of biological medicines and introduces a new drug design methods.Use of medicinal plants to treat diseases has a long history and now also in many developed countries is a main way for treatments.Iran has long been used in herbal remedies so that in ancient Iranian medical resources such as the writings of Avicenna (Ibn Sina) many sections devoted to this topic and plant diversity, with almost 9500 species of vascular plants in Iran is  more than the entire continent of Europe.

There are a huge number of researches about antimicrobial effects of Iranian plants derivatives(Aminnezhad et al., 2012; Hajimehdipoor et al., 2010;  Rahimifard et al., 2012- Rahimifard et al., 2014; Rahimifard et al., 2008; Rahimifard et al., 2015; Beiki et al., 2016; Amiri et al., 2016; Hafezan et al., 2016; Safarian et al., 2016; Mehrara et al., 2014).  Due to microbial resistance against antibiotics and side effects of chemical drugs, it is necessary to obtain new anti-bacterial compounds. Acinetobacter baumannii is one of the important pathogens in this respects.( Fournier et al.2006; Kim et al.,2004; Antunes et al., 2011)

Antimicrobial activities against some   species have been reported from the extracts and fraction of Dionysia revoluta Boiss( Ahani et al., 2016;Mashhadi et al., 2016; Rahimifard et al., 2016).  In the present study antimicrobial activities of methanolic and chloroformic and crude extracts against 50 clinical isolates of  Acinetobacter baumannii from the wound of  burned patients hospitalized at Motahari hospital in Tehran and Acinetobacter baumannii  ATCC 10572 were evaluated.

Methods and Materials

Plant Material and Preparation of Extracts

The samples of plant were collected from Hormozgan province of Iran. The plant were dried with rotary evaporator,  blended and extracts were prepared by standard methods of maceration. Methanolic extract of Dionysia revoluta Boiss were extracted with methanol 80%(1:10) by using maceration method for 4 days. After every 24 h, the mixture was filtered and new solvent was added to the plant powder. The extract were concentrated under reduced pressure to dryness and 8 concentrations of methanol, chloroform and water extract of Dionysia revoluta  ( 2000, 1000, 500, 250, 62.5,125, 31.25, 15.6 μg / ml)  were prepared.

Bacterial Isolation and Identification

In the present study 50 clinical isolates of Acinetobacter baumannii were used. All clinical strains were isolated from the Burning wound of patients hospitalized at Motahari hospital in Tehran. Primary isolation was performed on EMB(Merck) and also Blood agar base plates supplemented with 7% ship blood, after incubation at 37°C, with aerobic atmosphere , for 24 hours. Following primary selective isolation, Acinetobacter baumannii samples were identified by usual diagnostic procedures, i.e. according to colony morphology, gram staining and biochemical tests and Microgen identification kit GN A  and GN B.(Microgen Bioproducts)

Disk Diffusion Agar Method

Agar disk diffusion assay (Cup plate assay)was carried out on Muller-Hinton agar(Merck1.05437.0500)  by Kirby-Bauer disk diffusion susceptibility test protocol. Muller-Hinton agar was poured in sterile plates and plate’s surfaces were inoculated with approximately (1.5*10⁸ CFU/ml) equal to 0.5 McFarland turbidity of inoculum of  50 clinical isolates of  Acinetobacter baumannii and Acinetobacter baumannii  ATCC 10572 as Standard strain by sterile swab. The inoculum optical density (OD) had been adjusted between 0.08-0.13 in 620 nm in spectrophotometer. Standard blank disk with 6.4 mm diameter were put on plate (with an approximate distance of 19 mm . (Shokraei et al., 2014) 8 concentrations of methanol, chloroform and water extract of Dionysia revoluta  ( 2000, 1000, 500, 250, 62.5,125, 31.25, 15.6 μg / ml)  were prepared and 20 µl of each was poured on each blank disk and plates were incubated for 24 hours at 37°C with closed lid and aerobe conditions. Then the diameters of absence of growth were measured.

Well Diffusion Agar Method

Agar well diffusion assay was carried out on Muller-Hinton agar. Muller-Hinton agar (Merck1.05437.0500) was poured in sterile plates and plate’s surfaces were inoculated with approximately (1.5*10⁸ CFU/ ml) equal to 0.5 McFarland turbidity of Acinetobacter baumannii  strains by a sterile swab. The inoculum optical density (OD) had been adjusted between 0.08-0.13 in 620 nm in spectrophotometer. Wells were cut on plate by sterile Pasteur pipet (with an approximate distance of 19 mm). Wells were filled by 100 µl of  8 concentrations of methanol, chloroform and water extract of Dionysia revoluta  ( 2000, 1000, 500, 250, 62.5,125, 31.25, 15.6 μg / ml) and plate were incubated for 24 hours at 37°C with closed lid and aerobe conditions. The clear zone around wells then was recorded. (Barzavar et al., 2015)

Results

Table 1: Average inhibition zone diameter in millimeters in three times for different concentrations of methanol, chloroform and aqueous extracts of  Dionysia revoluta  on 16 of 50 strains of Acinetobacter baumannii in disc method (discs diameter  are 7 mm.)

NIZ: No Inhibition Zone/Resistant

According to the table all the plant extracts in concentrations less than 62.5 micrograms per milliliter has no effect on the growth of A. baumannii (50 clinical strains).

In general, the smallest diameter of inhibition was  in extracts of methanol and chloroform fraction  in a concentration of 62.5 micrograms per milliliter. And the aqueous fraction at a concentration of 2000 micrograms per milliliter has maximum inhibition zone which is equal to 15 mm.

Graph 1: Average inhibition zone diameter in millimeters in three times for different concentrations of methanol, chloroform and aqueous extract of  Dionysia revoluta  on 16 of 50 strains of Acinetobacter baumannii in disc method (discs diameter  are 7 mm.)   Click here to View Graph

Table 2: Average inhibition zone diameter in millimeters in three times for different concentrations of methanol, chloroform and water extract of Dionysia revoluta   on 16 of the 50 strains of Acinetobacter baumannii  in well method.

NIZ: No Inhibition Zone.Resistant

According to the table all the plant extracts in concentrations less than 62.5 micrograms per milliliter has no effect on Acinetobacter baumannii (50 clinical strains).

The smallest inhibition zone was for chloroform fraction  in a concentration of 125 micrograms in diameter ml (equal to 2.10 mm). And the aqueous fraction at a concentration of 2000 micrograms per milliliter had the greatest inhibition which is equal to to 16 mm.

Graph 2: Average inhibition zone diameter in millimeters in three times for different concentrations of methanol, chloroform and water extract of Dionysia revoluta   on 16 of the 50 strains of Acinetobacter baumannii  in well method.   Click here to View Graph

Table 3: Average inhibition zone diameter in millimeters in three times for the effect of different concentrations of methanol, chloroform and aqueous fractions extracts of   Dionysia revoluta on  the strains of Acinetobacter baumannii ATCC 10572  in disc method (discs diameter are7 mm).

NIZ: No Inhibition Zone.Resistant

According to table all the plant extracts in concentrations of less than 31.25  micrograms per milliliter has no effect on the growth of  standard A. baumannii strains ATCC 10572.

The smallest diameter of inhibition in the aqueous fraction at concentrations 31.25 micrograms per milliliter (3.13 mm) and then in chloroform fractions at  concentration of 125 micrograms per milliliter (7.13 mm) were observed and the aqueous fraction in a concentration 2000 micrograms per milliliter had the most inhibition zone (20 mm).

Graph 3: Average inhibition zone diameter in millimeters in three times for the effect of different concentrations of methanol, chloroform and aqueous fractions extracts of   Dionysia revoluta on  the strains of Acinetobacter baumannii ATCC 10572 as standard  in disc method (discs diameter are7 mm).   Click here to View Graph

It should be noted that the results in the well method on A. baumannii ATCC 10572 as standard  were also quite similar results were obtained in a disk method and as well as a negative control for each of the extracts of methanol, water and chloroform fractions,in  the entire process was ineffective.

Graph 4: Comparison of wells, disk methods  and standard strain at concentration 2000 μg . ml  and positive control.   Click here to View Graph

All three extracts had antibacterial effects on Acinetobacter baumannii.Water extract at 2000 mg/ml showed the maximum inhibition zone (15 mm).Well diffusion methods was more efficient method. Sixteen isolates were sensitive to all extracts and showed growth inhibition zone from 12 mm to 15 mm. We concluded that the antimicrobial activity of Dionysia revoluta Boiss against Acinetobacter baumannii is valuable, but further investigation especially in vivo studies are recommended before final conclusion.

Suggestions

As you know, historically, the most important infectious diseases are the main causes of death in humans. Today, many synthetic drugs are manufactured with antibacterial properties. Although these drugs can effect on micro organism, they have side effects for human. In addition, due to the indiscriminate use of chemical agents resistance to antimicrobial agents has been created. The use of plants with medicinal properties so recently expanded to deal with the bacteria. Although antibacterial and antifungal effects of plants that have been studied are too high, Still, there are plants among the plants that have long been used as a medicinal substance but their  biological effects have not been studied yet. In the meantime, Primrose families like Dionysia revoluta due to their antibacterial effects have been demonstrated in recent years are given priority.In vivo tests subsequently recommended in order supplying pharmaceutical products that can be used in the treatment of diseases.It is suggested to separate and purification the antibacterial and antifungal compounds in plant extracts and fractions that the main factor responsible for the antimicrobial activity be detected So maybe we can change the molecular structure of this compound with an effective antimicrobial products introduced. It is hoped that in future more research on the antimicrobial effects of this plant have been conducted on different bacterial species and by determining the antimicrobial active ingredients of the plant, separation, purification, formulation and  preparation of various dosage forms of that, is an excellent step for illnesses that are created by different bacterial species to be removed.

Acknowledgement

The author sincerely thanks the Sarv Saadat Laboratory complexes in West sarv, Saadat abad, Tehran for their financial support and  kind assistant and hard efforts.

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