1. INTRODUCTION:

The global increase in mortalities caused by cancer, is a serious threat facing the human race. According to the world health organization reports^[1], cancer is one of the leading causes of death globally. It is responsible for about 9.6 million deaths in 2018. Colorectal cancer comes second after Lung cancer as leading cause of cancer mortalities (862,000 deaths in 2018). On the other hand, female breast cancer ranks as the fifth leading cause of death (627,000 deaths) in 2018. Nowadays, majority of research efforts are currently devoted to screen, discover, and identify novel isolates exhibiting promising anticancer capabilities.

Actinomycetes are a family of mycelial bacteria famous as rich sources of effective classes of antibiotics, such as rifamycins, β-lactams, aminoglycosides, tetracyclines, glycopeptides, and macrolides^[2,3]. Moreover, actinomycetes exhibited other promising biological activities such as anticancer, antifungal, and immunosuppressant agents^[4-8].

Streptomyces is the most famous among actinomycetes genera. Thanks to its ability to produce almost two thirds of all isolated antibiotics and about 40% of all microbial metabolites, including variety of bioactive compounds involved in many medical, and agricultural applications, as well as exhibiting antifungal, antiviral, anti-tumoral, anti-hypertensives, or immuno-suppressants activities ^[7,9]. Over 850 species of Streptomyces have been validly described and published till now (http://www.bacterio.net/streptomyces.html)^[7,10].

The first isolated Streptomyces species was in 1940 from soil^[11,12]. This isolate exhibited potent antimicrobial activity by producing actinomycin (dactinomycin), which has other biological activities such as an anticancer drug which is approved by FDA under the name Cosmegen^[13]. This drug appears in the 19th WHO Model List of Essential Medicines^[14]as a therapy for Wilms tumor, rhabdomyosarcoma, and gestational trophoblastic neoplasia^[15]. The list of anticancer compounds originated from Streptomyces includes but not limited to: bleomycin, mitosanes, and anthracyclines which is a class including many effective anticancer compounds such as doxorubicin^[15]. Doxorubicin was firstly produced by Streptomyces peucetius in 1957^[16], and it is still used in chemotherapy treatment against different breast and lymphomas cancers^[14]. Many antibiotics were isolated from Streptomyces, such as streptothricin, streptomycin, grisein, fradicin, platensimycin, daptomycin, ribostamycin, fosfomycin, kanamycin, noviobiocin, vancomycin, cycloserine, lincomycin, virginiamycin, viomycin, nystatin, tetracycline, cephalosporins, chloramphenicol^[17-20]. The potency of Streptomyces species encourages continuous screening for novel isolates with promising biological activities. Hence, the aim of this study is to isolate and identify new actinomycetes isolates from novel sources showing potent anti-cancer activities.

2. MATERIALS AND METHODS:

2.1. Isolation of actinomycetes:

2.1.1. Sample collection:

Bat faeces and soil samples were collected from seven different sites (A, B, C, D, E, F, and G) inside El shekh Sayed cave which lies 44 Km east to Nile river. (Fig. 1) It is located in El Bayadya village at latitude 26° 57' 34.8"N and longitude 31° 27' 41.0" E, about 4 Km south of El Badari which is a famous archaeological site in Asyut Governorate, Upper Egypt.

The seven samples are A, and B: soils from the middle of the cave; C: sample from the wall of the cave; D: soil sample 25m inside the cave; E, G: soil containing bat faeces at the entrance of the cave; F: rhizosphere soil at the entrance of the cave. Samples were collected from 25 meter deep inside the cave, kept in sterilized bags and transferred in cool box (4°C) and processed within 24h.

2.1.2. Isolation and Purification of actinomycetes

One gram of soil samples from each site was placed into 9ml of sterile distilled water. Ten- fold serial dilutions were prepared from the mixed solution. Isolation was conducted from suitable dilution of the soil samples by spread over the surface of agar plates of starch-casein medium^[21] which contained (g/l): soluble starch, 10; K₂HPO₄, 2; KNO₃, 2; NaCl, 2; casein 0.3; MgSO₄.7H₂O, 0.05; FeSO₄.7H₂O, 0.01; anhydrous CaCO₃, 0.02; agar, 20 and pH 7.1. After incubation for five days at 28±2°C, the plates were checked for colonies growth and single colonies were picked-up and streaked onto the surface of agar plate of the same isolation medium, and allowed to grow for five days. A touch of the terminal colonial growth of a single separate colony was transferred to pure slants of starch-casein medium to be preserved in refrigerator by regular sub-culturing every two months.

2.2. Molecular identification of the selected isolate

The molecular identification of selected isolate using PCR and 16S rRNA sequencing was conducted as following:

2.2.1. Genomic DNA (gDNA) isolation and PCR amplification

A culture of the Streptomyces zaomyceticus AA1 grown at 28°C on starch-casein for 3 days was used for the preparation of genomic DNA (gDNA). Cells were collected by centrifugation (4,000 x g for 10 minutes), and total gDNA was extracted and purified using E.Z.N.A.® Bacterial DNA Kit (D3350-01, Omega BIO-TEK, USA) according to manufacturer protocol. The specific gene amplification was conducted using Dream Taq Green PCR Master Mix (2X) (K1081, Thermo Fisher, USA) according to manufacturer protocol using Creacon (Holland, Inc) Polymerase Chain Reaction (PCR) system cycler. Two universal primers targeting the 16S rRNA gene, namely: 27F (5’- AGAGTTTGATCMTGGCTCAG-3’), and 1492R (5’- TACGGYTACCTTGTTACGACTT-3’) were used in the amplification mixture. The reaction was initiated with denaturation at 94°C for 5 min. Next, 30 cycles of amplification were performed by denaturing for 1 min at 94°C, annealing the primer for 1 min at 42°C, and allowing elongation for 3 min at 72°C^[22]. 1.0% agarose solution was used during electrophoresis process in a multi SUB Mini, Mini Horizontal Electrophoresis System (Cleaver Scientific, USA). Specific DNA bands were eluted from agarose gel, and resultant PCR products were purified using E.Z.N.A. ®Gel Extraction Kit, (D2500-01, Omega BIO-TEK, USA). Sequence analysis was employed using the ABI PRISM® 3100 Genetic Analyzer (Micron-Corp., Korea).

2.2.2. Data analysis:

Gel documentation system (Geldoc-it, UVP, England), was applied for data analysis using Total lab analysis software, ww.totallab.com, (Ver.1.0.1). Aligned sequences were analyzed on NCBI website (http://www.ncbi.nlm.nih.gov/webcite) using BLAST to confirm their identity. Genetic distances and Multi Alignments were computed by Pairwise Distance method using ClusteralW software analysis (www.ClusteralW.com). The nucleotide sequences were also compared with Streptomyces isolates sequences available in the GenBank.

2.3. Fermentation and Extraction of secondary metabolites

Fermentation was performed using two nutritional broth media, starch-casein broth (of the same constituents as used for isolation with elimination of agar) and ISP2 broth medium which contained (in g/l): yeast extract, 4; malt extract, 10; dextrose, 4; pH 7.3^[21]. For each fermentation medium, two Erlenmeyer flasks (of 1liter capacity) were prepared. Each flask contained 500ml medium and inoculated with 5 ml spore suspension that was prepared by adding 5 ml of sterile tween-80 solution (0.1%) to each slant of the strain A501and spores were released using a sterile needle. The flasks were then incubated at 28±2°C and 150rpm for 5 days. The whole contents of each flak was transferred to Erlenmeyer flasks (of 2 liter capacity) and extracted twice by mixing with the same volume (500) ml of ethyl acetate (AnalR, UK) and allowed for shaking for two hours. The ethyl acetate extract was separated by separating funnel and the two extracts were evaporated till dryness using rotatory evaporator (Heidolph Rotary Evaporator, Schwabach, Germany) at temperature of 50°C.

2.4. GC-MS analysis of silylated metabolites:

Metabolites analysis was carried out as follows. Briefly 5ml 100% methanol was added on 100mg of finely powdered extract with sonication for 30 min using frequent shaking, followed by centrifugation at 12,000×g for 10 min to remove debris. 100µl of the methanolic extract was aliquoted in a screw-cap vials and left to evaporate under a nitrogen gas stream until complete dryness. For derivatization, 150µL of N-methyl-N-(trimethylsilyl)-tri fluoroacetamide (MSTFA) that was previously diluted 1:1% with an hydrous pyridine was added to the dried methanolic extract and incubated at 60 °C for 45 min prior to analysis using GC-MS^[23].

The GC-MS analysis of the extracts was performed at Department of Medicinal and Aromatic Plants Research, National Research Center with the following specifications, Instrument: a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp., USA), coupled with a Thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TR-5 MS column (30 m x 0.32mm i.d., 0.25μm film thickness). Analyses were carried out using helium as carrier gas at a flow rate of 1.0mL/min and a split ratio of 1:10 using the following temperature program: 60°C for 1 min; rising at 4.0 °C/min to 240°C and held for 1 min. The injector and detector were held at 210^oC. Diluted samples (1:10 hexane, v/ v) of 1μL of the mixtures were always injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450.

2.5. Effect of extracts on Human colorectal cancer (HCT116) and Human breast carcinoma tumor (MCF7) cell lines

2.5.1 Cell culture:

HCT116 colorectalcarcinoma, and MCF7 breast carcinoma human tumor cell lines were cultured in 95% humidity, 5% CO₂ and 37°C. HCT116 was maintained in McCoy’s 5A, while MCF7 in MEM media, supplemented with 10% fetal bovine serum and 1% antibiotic.

2.5.2. Cytotoxicity assay:

Acid phosphatase assay was conducted to evaluate cytotoxicity as described previously^[24]. Briefly, human colorectal cancer cell line (HCT116), and human breast carcinoma cell line (MCF7) were used by seeding 10000 cell per well in 96 well plates, left overnight till attach, and then treated with different extracts for three days. For one plate, a substrate solution was prepared where 20mg tablet of pNPP (Sigma; cat. no. N2765) was dissolved in 10ml buffer solution (0.1 M sodium acetate, 0.1% triton X-100, pH 5.0). Cell monolayers were washed with 250μl PBS. 100μl of pNPP substrate solution were added per well, then plates were incubated for 4 hours at 37°C. 10μl of 1N sodium hydroxide stop solution were added per well. Absorbance was measured directly at wavelength 405nm. All samples were tested in triplicates, and 0.5% DMSO was used as negative control and 50μM cisplatin was used as positive control. Extracts were tested at serial dilutions with final concentration of 200, 100, 50, and 25μg/ml. Percent cytotoxicity = [1-(D/S)] × 100, where D and S denote the optical density of drug and solvent treated wells, respectively.

2.6. DPPH radical scavenging activity assay (Antioxidant Activity):

The free radical scavenging activity of extracts was evaluated by using the 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay described by^[25]. Extracts were tested at final concentrations of 200, 100, 50, and 25μg/ml using 0.1mM DPPH dissolved in methanol. After incubation for 30 min in dark at room temperature, the absorbance was measured at 517nm. Ascorbic acid (vitamin C) was used as positive control at final concentrations of 20 µg/ml. The DPPH solutions treated with 0.5% DMSO used as a negative control. The DPPH scavenging activity of extracts was calculated according to the equation:

Percentage reduction = (1-(X/ av(NC)) ×100

Where x indicates the absorbance of fraction and av(NC) indicates the average absorbance of the negative control. EC50 values were calculated using probit analysis utilizing the SPSS computer program (SPSS for windows, statistical analysis software package/version 9 /1989 SPSS Inc., Chicago, USA).

Fig. 1El shekh Sayed bat cave, Asyut governorate, Egypt. i and ii: Location of the cave illustrated by Google maps; iii: entrance of the cave; iv: bats on the wall of the cave; v, and vi: bat faeces on soil.

3. RESULTS:

3.1. Isolation and molecular identification of actinomycetes:

Soil samples were collected from 7 sites inside the bat cave. The pH values of these samples were ranging from 5.5 to 6.5 (Data not shown). Isolation plates of samples from all sites showed reduced number or of actinomycetes. The only exception was in case of site G (representing soil sample containing bat faeces at the entrance of the bat cave) which showed several colonies of actinomycetes with special remarkable predominance of an isolate that is characterized by producing diffusible exo-pigment of yellowish color. This isolate was selected, purified, and was assigned as isolate AA1.Molecular identification of isolate AA1 using 16S rRNA sequencing confirmed that this isolate belongs to the genus Streptomyces. The sequence was deposited in the international GenBank under accession number MN567306. Phylogenetic tree was constructed based on 16S rRNA gene sequence (Fig. 2) showed that Streptomyces zaomyceticus strain AA1is closely related to Streptomyces zaomyceticus strain NRRLB-2038, and Streptomyces zaomyceticus strain NBRC 13348, and similarity of base sequence was more than 99%.

Fig. 2 Phyllogenetic tree of 16S rRNA sequence of the actinomycetes isolate Streptomyces zaomyceticus strain AA1with the sequences from NCBI.

3.2. GC –MS analysis of silylated metabolites:

Sialylation of the ethyl acetate extracts was conducted as a way to detect polar and non-polar compounds contained in those extracts. This was followed by performing GC-Ms analysis (Fig. 3 a, and b).

As shown in tables 1 and 2, the GC-Ms analysis of the silylation metabolites in the ethyl acetate extracts of isolate Streptomyces zaomyceticus strain AA1 revealed the presence of 22 compounds in each extract. Similar compounds were noticed between the two extracts such as α-Ketoglutaric acid; pentadecanoic acid; hexadecanoic acid (Palmitic Acid); 7,3',4' trimethoxy quercetin; 2-methyl Heptanedioic acid; and 3-Hydroxy butanoic acid. On the contrary, lactic acid; 2-methyl octanoate; 3-hydroxy-isovaleric acid; succinic acid; 3-hydroxy-2-cyclohexene-1- acetic acid; 2-(2-hydroxyphenyl)acetic acid; 2,3 dihydroxy butane; 2,2'bipyridine; N-Isopropyl-2-phenyl-6-propyl-4-pyridinamine; diisooctyl phthalate; maltose; trehalose; ethyl isoallocholate; dotriacontane; 2-bromo-octadecanal; and isochiapin B were uniquely observed in the ethyl acetate extract of isolate Streptomyces zaomyceticus strain AA1 growing on ISP-2 broth medium. On the other hand, cephaloridine; 2-methyloctanoicacid; glycerol; methyl-7-(2,4,6-trimethylphenyl)-5H-furo[2,3c] thiopyran-4-carboxylate; methylmalonic acid; 3-methylbut-2-en-1-yl methyl N (4-chlorophenyl) Dithiocarbonimidate; 2-hydroxy benzoic acid; ethyl-7-oxooctanoate; seco laurolitsine; tetradecanoic acid (Myristic Acid); 10-undecynoic acid; octadecanoic acid; and mono(2-ethylhexyl) phthalate were the compounds recorded in the ethyl acetate extract of isolate Streptomyces zaomyceticus strain AA1 growing on starch casein broth medium.

Table (1). Identified compounds of Streptomyces zaomyceticus strain AA1 extract cultivated at ISP-2 medium using GC-MS analysis

No	RT	Area %	Compound Name	Molecular Weight	Molecular Formula	SI
1	4.25	2.96	2-hydroxypropanoic acid (Lactic acid)	90	C₃H₆O₃	910
2	6.07	5.23	3-Hydroxy butanoic acid	104	C₄H₈O₃	851
3	6.54	3.96	2-methyl octanoate	214	C₁₃H₂₆O₂	614
4	6.99	4.1	3-Hydroxy-isovaleric acid	118	C₅H₁₀O₃	834
5	9.13	2.31	Butanedioic acid (Succinic acid)	118	C₄H₆O₄	813
6	10.27	3.76	2-Ketoglutaric acid	146	C₅H₆O₅	763
7	10.98	4.90	3-hydroxy-2-Cyclohexene-1- acetic acid	156	C₈H₁₂O₃	624
8	13.5	2.61	2-methyl Heptanedioic acid	174	C₈H₁₄O₄	648
9	13.64	2.4	2-(2-hydroxyphenyl) acetic acid	152	C₈H₈O₃	897
10	13.76	3.47	2,3 dihydroxy butane	90	C₄H₁₀O₂	632
11	18.27	2.39	2,2'Bipyridine	156	C₁₀H₈N₂	621
12	18.34	3.27	N-Isopropyl-2-phenyl-6-propyl-4-pyridinamine	254	C₁₇H₂₂N₂	631
13	19.08	3.17	Pentadecanoic acid	242	C₁₅H₃₀O₂	826
14	20.46	2.53	Hexadecanoic acid (Palmitic Acid)	256	C₁₆H₃₂O₂	821
15	27.08	22.36	Diisooctyl phthalate	390	C₂₄H₃₈O₄	943
16	28.29	2.76	Maltose	342	C₁₂H₂₂O₁₁	832
17	28.5	2.88	Trehalose	342	C₁₂H₂₂O₁₁	787
18	28.91	2.88	Ethyl isoallocholate	436	C₂₆H₄₄O₅	722
19	30.28	3.91	Dotriacontane	450	C₃₂H₆₆	730
20	30.47	3.33	2-bromo-Octadecanal	346	C₁₈H₃₅BrO	724
21	32.62	2.34	Isochiapin B	346	C₁₉H₂₂O₆	743
22	33.39	2.37	7,3',4' trimethoxy quercetin	344	C₁₈H₁₆O₇	738

Total identified % is 89.89 and SI ≥ 600

Table (2). Identified compounds of Streptomyces zaomyceticus strain AA1 extract cultivated at starch casein medium using GC-MS analysis

No	RT	Area %	Compound Name	Molecular Weight	Molecular Formula	SI
1	3.30	2.56	Cephaloridine	415	C₁₉H₁₇N₃O₄S₂	622
2	6.07	1.88	Hydroxy butanoic acid	104	C₄H₈O₃	879
3	6.54	1.83	2-Methyloctanoicacid	158	C₉H₁₈O₂	604
4	8.11	0.98	Glycerol	92	C₃H₈O₃	752
5	8.16	0.95	Methyl-7-(2,4,6-trimethylphenyl)-5H-furo[2,3c] thiopyran-4-carboxylate	314	C₁₈H₁₈O₃S	874
6	9.14	0.98	Methylmalonic Acid	118	C₄H₆O₄	685
7	9.32	1.61	3-Methylbut-2-en-1-yl Methyl N(4-chlorophenyl) Dithiocarbonimidate	285	C₁₃H₁₆ClNS₂	765
8	10.27	3.47	α-Ketoglutaric acid	146	C₅H₆O₅	758
9	12.65	1.86	2-hydroxy benzoic acid	138	C₇H₆O₃	883
10	13.50	3.19	Heptanedioic acid, 2-methyl	174	C₈H₁₄O₄	661
11	13.60	2.54	Ethyl-7-Oxooctanoate	186	C₁₀H₁₈O₃	657
12	13.76	0.95	seco Laurolitsine	313	C₁₈H₁₉NO₄	762
13	17.63	1.85	Tetradecanoic acid (Myristic Acid)	228	C₁₄H₂₈O₂	824
14	17.73	1.40	10-Undecynoic acid	182	C₁₁H₁₈O₂	689
15	19.07	1.49	n-Pentadecanoic acid	242	C₁₅H₃₀O₂	846
16	19.18	3.85	n-Pentadecanoic acid	242	C₁₅H₃₀O₂	800
17	20.45	6.82	Hexadecanoic acid (Palmitic acid)	256	C₁₆H₃₂O₂	923
18	20.7	0.94	Palmitelaidic acid	254	C₁₆H₃₀O₂	742
19	20.97	4.6	Hexadecanoic acid (Palmitic acid)	256	C₁₆H₃₂O₂	870
20	23.54	1.54	Octadecanoic Acid	284	C₁₈H₃₆O₂	733
21	27.08	50.74	Mono(2-ethylhexyl) phthalate	278	C₁₆H₂₂O₄	949
22	32.63	0.94	7,3',4' trimethoxy quercetin	344	C₁₈H₁₆O₇	734

Total identified % is 96.97 and SI ≥ 600

3.4. Antioxidant activity of the extract:

The hydrogen-donating or radical scavenging ability of the ethyl acetate extracts of Streptomyces zaomyceticus strain AA1 was investigated using DPPH as reagent. As shown in Fig 5, both extracts exhibited moderate antioxidant capabilities as a dose dependent inhibition of DPPH activity. The ethyl acetate extract of Streptomyces zaomyceticus strain AA1 cultivated on ISP 2 broth medium at concentration of 200µg/ml showed a higher DPPH scavenging % in comparison with the ethyl acetate extract of Streptomyces zaomyceticus strain AA1 cultivated on starch casein broth medium at the same concentration (53.5±3.4%, and 36.5±4.4%, respectively).

Fig. 5DPPH radical scavenging activity of the ethyl acetate extracts of Streptomyces zaomyceticus strain AA1 cultivated on starch casein broth medium (represented by closed circles); and cultivated on ISP2 broth medium (represented by closed triangles) at different concentrations. Values are presented as the means ± standard deviation (error bars) for three independent experiments.

4. DISCUSSION:

Actinomycetes are filamentous bacteria that are famous for their ability to produce variety of secondary metabolites of medicinal, biotechnological, agriculture, and industrial applications^[26]. Actinomycetes are the masters of antibiotics production. They are responsible for the production of about two thirds of the medically important antibiotics^[7,9]. Identification of novel isolates or isolates exhibiting potent biological activities is of great interest. Hence, isolation of actinomycetes especially from remote area is attracting attention all over the world. A remote bat cave located in Asyut governorate, Egypt has been chosen as a source to isolate actinomycetes. Out of the resulted isolates, one was predominant and characterized by producing diffusible exo-pigment of yellowish color. This isolate was identified by 16s rRNA as Streptomyces zaomyceticus strain AA1 and its sequence was deposited in the international gene bank under the accession number MN567306.Streptomyces species are the producers of around half of all known antibiotics^[27].

Streptomyces zaomyceticus strain AA1 was investigated for potential biological activities using two different growth media, ISP2, and starch casein. Silylation process was conducted in order to detect and identify as much polar and non-polar compounds as possible by GC-MS analysis. As a result, both extracts revealed profound effect of culture media on qualitative and quantitative profiling of metabolites produced (Tables 1,2). Only eight metabolic products of total 44 compounds (18.18%) were similarly identified in both extracts of the two culture media. On the other side, remaining 36 metabolites (81.82%) were different in both extracts of the two used medium. Additionally, the eight common metabolic products (3-hydroxy butanoic acid, 2-methyl octanoate, 2-ketoglutaric acid, 2-methyl heptanedioic acid, 2,3 dihydroxy butane, pentadecanoic acid, palmitic acid and diisooctyl phthalate) showed different quantitative distribution in the two culture media. Changing composition of carbon, nitrogen, as well as phosphorus sources have been previously reported to affect secondary metabolites in actinomycetes^[28]. Interestingly, most of the identified compounds by the help of GC-MS were of biological importance.

Bipyridyl (2,2'Bipyridine) is a famous herbicide^[29] and antifungal agent^[30]; octadecanoic acid (known also as stearic acid)appears to be as effective as oleic acid in lowering plasma cholesterol levels^[31]; 2-Hydroxyphenylacetic acid is used as an important intermediate in the pharmaceutical industry to prepare many bioactive compounds such as antihypertensive agents^[32]. Succinic acid has a wide range of applications in food and pharmaceutical industries. Many studies have reported the antimicrobial, antioxidant, and anti-larval activities of succinic acid^[33,34]. Furthermore, it acts as cerebroprotective, cardioprotective, immunotropic and stress protective agent, and has possible role in treatment of some mitochondrial diseases in children^[34,35]. Trehalose is a multifunctional molecule that is used in many industries^[36]. Moreover, trehalose expression in mammalian cells contributes in conferring desiccation tolerance on human cells^[37]; 10-Undecynoic acid is a highly selective irreversible inhibitor of hepatic ω- and ω-1-hydroxylases^[38]. Furthermore, it has antimicrobial activities^[39]. The Sesquiterpen lactone (Isochiapin B) is known for its anti-insect, antimicrobial, antioxidant, and anticancer activities^[40-42]. Lactic acid has a famous antimicrobial activities against bacteria and fungi^[43], and many studies have previously reported the antibacterial activity of lactic acid against Escherichia coli, Listeria monocytogenes, Salmonella gaminara^[44], Salmonella enteritidis^[45], Staphylococcus aureus^[46], also against resistant clinical isolates of Pseudomonas aeruginosa^[47] and spore-forming bacteria^[48]. Moreover, several studies suggested that lactic acid is more efficient antibacterial agent than acetic, citric and propionic acid^[49-51]. On the other hand, cephaloridine was observed in the silylated extract of Streptomyces zaomyceticus strain AA1growing on starch casein broth medium was resulting from the silylation process of cephalosporin indicating that the isolate Streptomyces zaomyceticus strain AA1 is a cephalosporin producer. Investigating the in vitro anticancerpotential of both ethyl acetate extracts revealed that the starch casein extract exhibited higher cytotoxic activity (94.03±0.1%) against human colorectal cancer cell line HCT116 using 200μg/ml of the extract, while the ISP 2 originated extract exerted higher cytotoxic activity (97.07±0.8%) against human breast cancer cell line MCF7 (Fig. 4a, and b). As far as we know, this is the first reporting for the anticancer capabilities of extracts from the species Streptomyces zaomyceticus, majority of published studies were reporting the antimicrobial activities of Streptomyces zaomyceticus only^[52,53].

The anticancer capacities of both extracts can be attributed to the presence of some compounds such as 3-hydroxy butanoic acid, and 2-ketoglutaric acid which were commonly detected in both extracts, and which have been previously reported to have anticancer activity^[^54,55]. Moreover, the compound, isochiapin B was identified from the ethyl acetate extract of Streptomyces zaomyceticus strain AA1 grown on ISP-2 broth medium has been also reported to have anticancer action^[41].

On the other hand, both ethyl acetate extracts of Streptomyces zaomyceticus strain AA1 exhibited a moderate antioxidant activity (Fig 5a, and b), as a dose dependent inhibition of DPPH activity. The ethyl acetate extract of Streptomyces zaomyceticus strain AA1 cultivated on ISP 2 broth medium at on centration of 200 µg/ml showed a higher DPPH scavenging activity (53.5± 3.4%) in comparison with the ethyl acetate extract of Streptomyces zaomyceticus strain AA1 cultivated on starch casein broth medium at the same concentration (36.5±4.4%). The presence of Succinic acid, as well as isochiapin B which are known for their antioxidant activities could explain the increased antioxidant activity of the in ISP 2 originated extract^[56,57]. Many studies have previously discussed the antioxidant activities of actinomycetes members^[58-61]. In this study, identification of a new actinomycetes isolate was conducted. Screening and isolation of actinomycetes for the purpose of investigating their potential biological activities is attracting researchers attention in past, present and future as previously reported^[62-67].

5. CONCLUSION:

Searching for isolates having promising biological activities especially against cancer is target of current researches all over the world. In this study, it was emphasized that screening for microbes in remote areas will always result in discovery of promising isolates with remarkable activities. Further in vivo studies is required to complete investigating potentials of the promising isolate, Streptomyces zaomyceticus strain AA1.

6. ACKNOWLEDGMENT:

The authors are thankful for Professor Stig Linder, Karolinska Institute, Sweden, for providing the cell lines. Also, authors are grateful for Dr. Ahmed I. Marzouk, Delta scientific consultancy center for sequencing service.

7. CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

8. FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

9. REFERENCES:

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Received on 07.11.2019 Modified on 13.12.2019

Research J. Pharm. and Tech. 2020; 13(7): 3072-3080.

DOI: 10.5958/0974-360X.2020.00545.4