Introduction

With the continuing search for novel natural products, it has become evident that the rate of re-isolation of known compounds has increased; therefore new microbial habitats need to be examined. One relatively overlooked but biologically important niche is the inner tissues of higher plants (Qin et al. 2009). All plants retain a microbiota (Bascom-Slack et al. 2009) and organisms that establish an endosymbiotic relationship with plants are defined as endophytes. Actinomycetes are saprophytes well known for producing a wide range of secondary metabolites. However, very little information is available to us regarding the biodiversity, tissue distribution, and biosynthetic potential of endophytic actinomycetes residing in wild, native plants (Janso and Carter 2010).

The Asteraceae family includes some of the most valued and oldest medicinal plants (Jeschke et al. 2009). Ageratum conyzoides L. is widely distributed all over Pakistan (Singh et al. 2012) and locally known as ‘Neeli Booti’ (Zafar et al. 2006). It has been used in traditional medicine for the treatment of innumerous disorders. However, no report of the in vitro anticancer activity of the plant has been evaluated or published (Adebayo et al. 2010).

Sonchus asper is another important herbal plant used as an alternative in the treatment of various diseases. It is a rich source of phenolic compounds, flavonoids, carotenoids, sesquiterpene lactones glycosides, ascorbic acid, and ω-3 fatty acids. (Khan et al. 2010).

The aim of the study was to explore the diversity and analyze the endophytic actinomycetes isolated from parts of medicinal plants belonging to Asteraceae family for specific biological activities. Actinomycetes were isolated from the medicinal plants from several of its natural habitats in Pakistan. Keeping in mind there is a lack of data on the antioxidant and cytotoxicity studies of endophytic actinomycetes from these plants therefore this study provides new and useful information.

Materials and methods

Plant material and collection site

Healthy plants of Parthenium hysterophorus were collected from the agricultural lands at the department of microbiology and molecular genetics (MMG) located at the University of the Punjab, Lahore. Ageratum conyzoides, Sonchus oleraceus, Sonchus asper and Hieracium canadense were collected from shady, moist areas around the department (31.49°N, 74.29°E, elevation ~210 m).

Sample treatment and isolation

Each plant sample was thoroughly washed in running tap water to remove soil particles and adhered epiphytes. After drying in sterile conditions, tissues surfaces were divided into 0.5 cm segments and surface sterilized by the five step sequential process as described by Tanvir et al. (2013) and plated.

Test organisms and antibiosis assay

For detection of antimicrobial activity, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 706003), Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas, Enterobacter, Bacillus, Candida tropicalis, and Chlorella vulgaris were used. The minimum inhibitory concentration (MIC) of the extracts obtained from endophytic actinomycetes were tested by broth dilution method as described by Andrews (2001). Extraction of fermentation broth of endophytic strains was done with ethyl acetate which was dried on a rotary evaporator to yield crude extracts.

Brine shrimp lethality test

For brine shrimp cytotoxicity assay, the extracts were dried and dissolved in DMSO to obtain the desired concentrations. The final concentration of DMSO in the assay volume was kept at 1 % (v/v) to prevent possible false effects originating from DMSO toxicity. Actinomycin D was used a positive control and artificial sea water was used as a negative control for the assay. Toxicity of the crude extracts was tested at 10, 50, 100, 150, 500, 1,000 μg/ml in seawater containing 1 % (v/v) DMSO. The mortality rate M was calculated using the formula:

$$ {\text{M}} = \left( {\frac{{\left( {{\text{A}} - {\text{B}} - {\text{N}}} \right)}}{{\left( {{\text{G}} - {\text{N}}} \right)}}} \right) \times 100 $$

where M is the percent of the dead nauplii after 24 h; A is the number of the dead nauplii after 24 h; B is the average number of the dead nauplii in the blind samples after 24 h; N is the number of dead nauplii before starting the test; G is the total number of nauplii.

Cytotoxicity was considered significant if the LC50 value was less than 20–30 μg/ml (Geran et al. 1972).

Free radical scavenging activity assay using diphenylpicrylhydrazyl (DPPH)

Antioxidant activity was tested according to the procedure by Pajero et al. (2000) with slight modifications. Antioxidant activity of the crude extracts was tested at 100, 500, 1,000, 5,000 and 10,000 μg DPPH/ml dissolved in methanol. An ascorbate standard was used as a positive control and DPPH stock was used as a negative control. The DPPH radical scavenging activity of the sample was calculated as:

$$ \left[ {{\text{Abs}}_{\text{control}} {-}{\text{ Abs}}_{\text{sample}} } \right] \, /{\text{ Ab}}_{\text{sample}} \times 100 $$

The antioxidant activity was expressed in terms of EC50, which was defined as the dilution of the crude extract needed to decrease initial DPPH chemilumininescence intensity by 50 %.

Statistical analysis

All experiments were performed in triplicates and results are presented as mean values. The calculations for LC50 were done through probit regression analysis and the determination of EC50 was done through dose response curve. Analysis was done using statistical package SPSS version 20.0 (SPSS, USA).

Results and discussion

A variety of plant species have been explored for the isolation of endophytic actinobacteria ranging from different woody tree species, ferns, club mosses even crop plants, like rice, wheat, carrots, potato, tomato and citrus (Qin et al. 2011a, b). However, there are few reports on bioactive compounds from actinomycetes of such origin (Radhakrishnan et al. 2010). This situation encouraged us to explore and investigate the endophytic actinomycetes community of medicinal plants in family Asteraceae. Therefore this study is one of its kind to describe the biodiversity and bioactivities of endophytic actinomycetes associated with the plants described.

Our study revealed that the tissues of these plants harbor a variety of endophytic actinomycetes. A total of 182 isolates were obtained from the stem, root and leaf of the plants. Most isolates were identified as Streptomyces spp. after cultural characterization. Of these 182 isolates, a majority were recorded from the roots (n = 127, 69.7 %) then shoots (n = 32, 17.5 %) and leaves (n = 23, 12.6 %). The Streptomyces genus was dominant (n = 96, 52.7 % of all isolates), followed by Micromonospora (n = 3, 1.5 %), Amycolatopsis (n = 2, 1 %) and Pseudonocardia, Nocardia (n = 1, 0.5 %) (Table S1).

Among the isolates, various subgroups were identified and among Streptomyces spp., the subgroup Streptomyces rochei (n = 3, 7.1 %) was also isolated. Apart from our previous study (Tanvir et al. 2013), no other study has reported its isolation from Asteraceae plants. The roots of Asteraceae plants harbored besides Streptomyces spp., other genera like Amycolatopsis, Nocardia, Pseudonocardia, and Micromonospora. There are reports of isolation of Micromonospora tulbaghia sp. nov., from the leaves of Tulbaghia violacea (Kirby and Meyers 2010), and Pseudonocardia sichuanensis sp. nov., from the root of Jatropha curcas L. (Qin et al. 2011a, b).

Considering these findings, it is apparent that a selected group of actinomycetes namely Streptomyces, Amycolatopsis, Nocardia, Pseudonocardia, and Micromonospora are able to form endophytic associations with a wide range of plant hosts.

Eighty-six of the isolates (47.2 %) showed growth inhibitory activity against pathogenic strains of microorganisms and among them 36 (41.8 %) isolates had significant broad spectrum antimicrobial activity while 25 (29.0 %) showed strong bioactivity. The isolates RT 6, 13, 18, 36, 49, 56, 67 and AGRS8, SORS80 (all isolated from the roots) were further characterized by 16S rRNA sequencing (Table S2).

Streptomyces strains, such as RT 46, 49, 54 and 63, exhibited major MIC values ranging from 4–32 μg/ml against all nosocomial pathogens (Table 1). One of the isolate AGRS8 with 99 % homology to Streptomyces albovinaceus showed the most significant MIC value of 2 μg/ml against all multidrug resistant isolates whereas another strain AGLS2 identified as Pseudonocardia carboxydivorans and SORS80 identified as Amycolatopsis japonicum also showed 4–8 μg/ml MIC values against all test organisms as shown in Table 2.

Table 1 MIC of the endophytic actinomycetes isolates against nosocomial pathogens
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Table 2 Minimal inhibitory concentration (MIC) for determining the antimicrobial, antifungal, and antialgal potential of the endophytic actinomycetes from Asteraceae plants
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Exposure of brine shrimp to extracts also revealed interesting results: in case of strains RT 13, 50, 59 and 67 the percentage mortality after 24 h ranged from 96.6 % at 100 μg/ml to 100 % at 500, 1 and 5 mg/ml. A high mortality rate was also observed in the isolates RT 18, 36 and 53 which gave 100 % mortality at 100 μg/ml which was the lowest concentration for these extracts tested (Fig. S1). The LC50 value for the extracts was very potent at 20 μg/ml, which is considered extremely toxic when compared to values provided by Geran et al. (1972). Overall, 15 of the 33 isolates selected for cytotoxicity showed an extremely potent cytotoxicity value of LC50 10 μg/ml whereas two isolates showed LC50 values between 23 and 50 μg/ml which is also very toxic (Table 3).

Table 3 Cytotoxicity results of endophytic actinomycetes from Parthenium hysterophorus, Ageratum conyzoides, Sonchus oleraceus and Hieracium canadense after 24 h
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It is evident from our study that the endophytic actinomycetes residing in the plants of Asteraceae family have significant capability of producing cytotoxic compounds. Previous reports also support our results with novel antibiotics such as coronamycin from endophytic Streptomyces sp. with cytotoxicity of IC50 5–10 μg ml (Ezra et al. 2004). It establishes the fact that endophytes are excellent source for the isolation of novel cytotoxic compounds.

In light of the previous reports regarding the antioxidant activity for the Streptomyces, the activity for endophytic actinomycetes was quantified using the DPPH free-radical scavenging activity assay. As compared to the ascorbate standard for which the EC50 was 0.34 μg/ml, the EC50 of all of the actinomycetes strains gave impressive results. The EC50 of the isolates SORS64b, SORS124, AGRS16, AGLS2, AGRS19, gave EC50 values of 0.55, 0.57, 0.62, 0.65, and 0.67 μg/ml, respectively, with 0.552 and 0.567 μg/ml being the highest EC50 value (Table 4). The remaining isolates also gave good results with EC50 value of 0.7 ± 2 μg/ml when compared to the ascorbate standard. According to our study, the antioxidant activity of the crude extracts increased with the increase in concentration which is similar to the results of Kekuda et al. (2010) who reported that with higher concentration the scavenging activity of the Streptomyces was also increased.

Table 4 Total antioxidant activity of different concentrations of endophytic actinomycetes
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Summary

This study suggests that the Asteraceae plant family harbors endophytic actinomycetes that produce a wide range of novel natural products with pharmaceutical potential. As such to the best of our knowledge, there is a lack of data on exploring the diversity of endophytic actinomycetes residing in the plant tissue of Asteraceae plant family, therefore this study not only describes an untapped environment but also highlights the potential usage of these isolates in biotechnological, medical and pharmaceutical applications.