IMMOBILIZATION AND APPLICATION OF PARTIAL PURIFIED LOVASTATIN PRODUCED FROM LOCAL ISOLATE ASPERGILLUS TERREUS A50 USING SOLID STATE FERMENTATION

This work was designed to study the free and immobilized partial purified lovastatin in various applications. The results of HMG-CoA reductase inhibition showed enzyme inhibition at 10 mM of standard and partial purified lovastatin with specific activity 0.056 and 0.062 U/mg protein respectively, compared with specific activity 0.277 U/mg protein without inhibitor. The results of the thermal stability and storage time on lovastatin for inhibition of HMG-CoA reductase demonstrated that the standard and partial purified lovastatin were stabled in temperatures between 20-40 oC, then the stability begun to decrease at 45 oC, while lovastatin was stable in storage time between 18 hours, then the stability begun to decrease after ten hours at 40 oC. The results of MIC for lovastatin were demonstrated that most tested concentration were showed antibacterial activity of free and immobilized partial purified lovastatin against Candida albicans, Escherichia coli, and Staphylococcus aureus with MIC values ranging from 15 to 75 μg/ml. Whereas the results of minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) showed that C. albicans, E. coli, and S. aureus had no growth with concentration ranging from 55 to 75, 55 to 75, and 30 to 75 μg/ml, respectively. As well as the results of the cytotoxic impact using MTT experiment indicated that partial purified lovastatin caused a reduction in cells viability (p ≤ 0.05) at a dose-dependent manner on MCF-7 cell lines, with a calculating IC50 of 138.1 μg/ml, compare with normal cell line (WRL 68 Cell Line) at IC50 of 198.7 μg/ml. Keyword: silver nanoparticles, HMG-CoA reductase activity, antimicrobial activity, anticancer activity. ةيقارعلا ةيعارزلا مولعلا ةلجم 2021 : 52 ) 2 :) 377 391 زيزعو يدعاسلا رطفل ةيلحملا هلزعلا نم جتنملاو اَيئزج ىقنملا نيتاتسافوللا قيبطتو دييقت Aspergillus terreus A50 تا رمخت مادختساب ةبلصلا ةلاحلا زيزع معنم يزاغ يدعاسلا كشر رابج يلع روتكد ذاتسا سردم ةيئايحلاا تاينقتلا مسق / علا ةيلك مول / دادغب ةعماج صلختسملا ممص لا لمع يلاحلا ل نيتاتسافوللا ةسا رد يئزج ىقنملا َا رحلا و ديقملا فلتخم يف تاقيبطتلا . طيبثت جئاتن ترهظأ ميزنا HMG-CoA Reductase نأ ميزنلإا طبث دنع 10 لم ي رلوم نيتاتسافوللا نم و يسايقلا يئزج ىقنملا َا عم ةيعون ةيلاعف 0,056 و 0,062 ةدحو غلم/ ار ةنراقم ، يلاوتلا ىلع نيتورب م عم ميزنلال ةيعونلا ةيلاعفلا 0,277 ةدحو / ما رغلم نيتورب و .طبثم نودب امك هظأ اقوا ريثأتو يرا رحلا تابثلا جئاتن تر فلتخملا نيزختلا ت ة ىلع طيبثتل نيتاتسافوللا ميزنا HMG-CoA Reductase يئزج ىقنملا نيتاتسافوللا نأ اَ م ناك يسايقلاو نيب حوا رتت ةرا رح تاجرد يف رقتس 20 40 ةجرد ضفخني تابثلا أدب مث ، ةيوئم دنع 45 ةجرد ةيوئم ، امنيب ناك ريثأت تقو نيزختلا عونتملا ىلع لا نيب نيزختلا تقو يف ا رقتسم نيتاتسافول 1 8 ضفخني تابثلا أدب مث ، تاعاس دعب رشع دنع تاعاس 40 .ةيوئم ةجرد دحلا جئاتن ترهظأ ( طبثملا زيكرتلا نم ىندلأا MIC ) نيتاتسافولل مظعم نأ رتلا ا لا زيك ت هرابتخا مت ي ا رهظأ ت هداضم ةيلاعف تابوركياملل مادختساب يئزج ىقنملا نيتاتسافوللا َا لاو رحلا ديقم دض ةريمخ Candida albicans ، و Escherichia coli و Staphylococcus aureus ميق عم MIC نم حوا رتت 15 ىلإ 75 لم /ما رغوركيم رتلي نم ىندلأا دحلا جئاتن ترهظأ امنيب . لا زيكرت لتاقلا ايرتكبلل ( MBC نم ىندلأا دحلاو ) لا زيكرت ل لتاقلا ( تايرطفل MFC نأ ) C.albicans و E.coli و S.aureus مل ت ظ يا ره ومن يف لا ا رت زيك يتلا ت نم حوا رت 55 ىلإ 75 , و 55 ىلإ 75 , و 30 ىلإ 75 لم/ما رغوركيم رتلي لا ريثأتلا جئاتن تراشأ .يلاوتلا ىلع ، ىلع ماس ا ايلاخل ةبرجت مادختساب MTT ىقنملا نيتاتسافوللا نأ ىلإ يئزج َا ضافخنا يف ببست ايلاخلا ةيويح ( (p ≤ 0.05 ةعرجلا ىلع دمتعت ةقيرطب و ايلاخ طوطخ ىلع MCF-7 باسح عم ، IC50 يلاوح 138,1 لم /ما رغوركيم رتلي ( يداعلا ةيلخلا طخ عم ةنراقم ، ةيلخلا طخ WRL 68 دنع ) IC50 يلاوح 198,7 لم/ما رغوركيم رتلي . تاملكلا :ةيحاتفملا ةيونانلا ةضفلا تائيزج ميزنا ، HMG-CoA Reductase .ناطرسلل ةداضملا ةيلاعفلا ،تابوركيملل ةداضملا ةيلاعفلا , Received:10/3/2020, Accepted:21/6/2020 Iraqi Journal of Agricultural Sciences –2021:52(2):377-391 Al-Sa'ady & Aziz 378 INTRODUCTION Lovastatin is polyketide components, created or produce via certain fungi during their secondary metabolism. Lovastatin, also known as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors, are a class of lipid-lowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. The statins act as competitive inhibitors for HMG-CoA reductase, a rate constraining advance of cholesterol biosynthesis (2). Statins hinder the change of HMG-CoA to mevalonic acid in the mevalonate pathway (18). Production of lovastatin by submerged fermentation and solid state fermentation has been widely investigated and commonly, filamentous fungi exhibit tremendous potentiality (7). Aspergillus terreus is a filamentous ascomycota, which is significant provenance for generation of lovastatin (6). The immobilization methods are used for the binding of cells, organelles, compounds, proteins (3), or different materials onto a strong help, into a strong lattice or held by a membrane, so as to build their stability and make conceivable their rehashed or proceeded with employ (16). Silver particles having fine or ultrafine sizes have attracted scientific interest because of their unusual properties compared to bulk metal (19). Colloidal particles because of their quantum size effects and surface effects reveal excellent electrical conductivity, catalytical activity, chemical stability and antimicrobial activity (14). Interestingly, some studies have demonstrated an antimicrobial potential for statins against different bacterial species. For example, simvastatin was able to inhibit host-cell invasion and Staphylococcus aureus growth. In addition, lovastatin, atorvastatin, simvastatin and rosuvastatin showed activity against several reference bacteria, yeasts and clinical isolates (11). Lovastatin and associated components indicated wonder impacts on tumor cells however site of activity and mechanism of activity is ineffectively comprehended. Concentrate done via Xiangli et.al. uncovered anti-proliferative impacts of lovastatin on malignant cells. The experiment was performed on the tumor in human glioblastoma cells and decrease in the malignancy was showed by lovastatin through inactivation of RAS farsonylation (29). The purpose of this study was to immobilization of lovastatin produced from local isolate Aspergillus terreus A50 using solid state fermentation with nano-silver and used of lovastatin in various applications. MATERIALS AND METHODS Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Nutrient agar, Nutrient broth from Hi-media, India. HMG-CoA Reductase activity kit (colorimetric) from abcam company. Sodium Acetate (CH3COONa), Sodium Hydroxide (NaOH), Ethanol 95%, methanol, ethyl acetate, trifluroacetic acid and other materials from BDH, England. Lovastatin production Collection of seventy three local fungal isolates from different areas, and screening for lovastatin production. The local isolate Aspergillus terreus A50 was best isolate for lovastatin production through submerged fermentation. The optimum conditions for lovastatin production by local isolate A.terreus A50 were used solid state fermentation (SSF) with media contain wheat bran and oat bran (1:1 w:w), sodium acetate, moisture ratio (1.2:1 v:w), pH (7), incubation temperature 30 °C and incubation period (6 days). Local isolate of A.terreus A50 was cultured on potato dextrose agar. Then lovastatin production from this isolate were performed by using a medium mention above and the growth was elicited with 1 ml (1x10 6 cells/ml)/5gm media of S.cerevisiae after 48 hours of culture. Extraction of lovastatin After the end of SSF operation, the concentration of lovastatin was measured. First, the culture was extracted in 250 ml Erlenmeyer's flask with ethyl acetate (pH 3.0). The mixtures was then incubated in rotating shaker with 140 rpm at 28 oC for 2 h. Next, filtration was done using Whatman filter paper (No. 1) for separation of the biomass from the filtrate. Then stored the supernatant in glass bottles at 4 oC until use for additional examination as crude extraction (26). Estimation and purification of lovastatin Lovastatin was estimated based on the method described by Mielcarek et al. (21) as follows: One ml of trifluroacetic acid (1%) was mixed with one ml of the supernatant and incubated for 10 min. A 0.5 ml from these mixture was diluted 10 times with methanol and its absorbIraqi Journal of Agricultural Sciences –2021:52(2):377-391 Al-Sa'ady & Aziz 379 ance was estimated at 238 nm using UVVisible Spectrophotometer. The lovastatin concentration in the sample was determined by plotting the O.D values on standard chart. Three ml of mixture from 0.5 ml trifluroacetic acid (1%) and 2.5 ml methanol were used as control. The purification of lovastatin produced by local isolate A.terreus A50 was performed by silica gel column chromatography (using silica gel column (67-1.5 cm) (60-120 Mesh) eluted with chloromethane :ethyl acetate (70:30 v:v), 3 ml for each fraction with flow rate 20 ml/ hour). Effects of partial purified lovastatin on HMG-CoA Reductase activity The study of the partial purified lovastatin effect on HMG-CoA reductase activity was performed by using kit (colorimetric) from abcam company. This experiment was achieved using 10 mM of standard, partial purified lovastatin, and atorvastatin (present in kit as standard inhibitor) . The enzyme was separately incubated with each inhibitor at 37 oC each 2 min for 10 min. The the percentage (%) of enzyme inhibition was estimated according to the producer of kit (colorimetric) from abcam company. The enzyme inhibition was estimated according to the equation: Inhibition %={[(−∆A 340nm/ΔT (Enzyme)) – (−∆A 340nm/ΔT (Enzyme+Inhibitor)] / −∆A 340nm/ ΔT (Enzyme) }X 100. A= Absorbance, T= Time Thermal stability of lovastatin for HMGCoA reductase inhibition: A 100 μl of standard and partial purified lovastatin separately were incubated in water bath at different temperature degrees (20, 25, 30, 35, 40, 45, 50, 55, and 60) OC for 30 min, then the test tubes containing the lovastatin were transferred directly to cold water. The impacts of inhibitors on HMG-CoA Reductase activity were determined by using 5 μl (10 mM) of standard and partial purified lovastatin. The enzyme was separately incubated with inhibitors at 37 oC each 2 min for 10 min. The enzyme inhibition % was estimated according to the same previous equation. Effect of different storage time of partial purified lovastatin on HMG-CoA reductase inhibition: A 100 μl of standard and partial purified lovastatin separately were incubated in water bath at 40 OC for different time 2, 4, 6, 8, 10, 24, 48 hours. The test tubes containing the lovastatin were then transferred directly to cold water bath. The effects of inhibitors on HMG-CoA Reductase activity were investigated by using 5 μl (10 mM) of standard and partial purified lovastatin. The enzyme was separately incubated with inhibitors at 37 oC each 2 min for 10 min. The enzyme inhibition % was estimated according to the same previous equation. Immobilization of lovastatin (Binding Nanosilver with lovastatin): Lovastatin was suspended in deionizing water and sonicated for 15 min. Next, 2 ml of lovastatin suspension (0.5 mg/ml) was transferred to tube and mixed with 2 ml of standard silver nanoparticles of concentration 0.7 mg/ml. After incubation for 24 hours the solutions were centrifuged 10000 rpm, for 10 minutes at room temperature, the supernatant was transferred to tube and ignores. While the precipitate was washed for four times, then were subjected to complete analysis via ultraviolet-visible spectrophotometry and ATR-FTIR analysis, for detect the binding between lovastatin and standard silver nanoparticles (28). Lovastatin applications Antimicrobial activity of lovastatin: The bacterial isolates were obtained from department of Biotechnology/College of Science/ University of Baghdad, bacteria were: Grampositive: Staphylococcus aureus and Enterococcus faecalis, Gram-negative: Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa and the yeast: Candida albicans. The bacteria and yeast were sub-cultured on nutrient broth and incubated at 37°C, while yeast in 28 oC for 48 hours and then kept in the refrigerator for next experiments. The various concentrations of partial purified lovastatin were prepared using distilled water as solvent, concentration included 10, 50, and 75 μg/ml. The antibiotic (metronidazole) was prepared via distilled water with 50 μg/ml concentration. The metronidazole was used as control (1). Determination of lovastatin activity by agar diffusion method: According to Obeidat et al. (23), petri-dish plates contained of MuellerHinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with an overnight culture of Iraqi Journal of Agricultural Sciences –2021:52(2):377-391 Al-Sa'ady & Aziz 380 indicators strains. Five wells (5 mm diameter) were made into the (two plates) agar via corkborer and three wells were loading with 0.1 ml of the partial purified lovastatin with different concentration (10, 50, and 75 μg/ml) and standard lovastatin with concentration of 75 μg/ml, as well as the metronidazole and distilled water were used as control. The inoculums volume was balanced in order to convey last inoculums of around 7×10 6 cells/ml, cells enumeration was doing by a hemocytometer. Incubation of bacteria were performed at 37 oC and of yeast at 28 oC for 24 hours. The assessment of antibacterial and antifungal activity was based on diameter measurement of the inhibition zone formed around the well. Antimicrobial activity of immobilized lovastatin: Petri-dish plates of Mueller-Hinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with old culture (7×10 6 cells/ml) of same microbial strains that mention above. Five wells (5 mm diameter) were made into (two plates) the agar via cork-borer and 0.1 ml of different solutions were applied in each well, these solution including the 75 μg/ml of standard lovastatin, partial purified lovastatin, immobilized lovastatin, nano-silver, and distilled water as control. The assessment of antibacterial and antifungal activity was based on diameter measurement of the inhibition zone formed around the well (23). Determination of minimum inhibition concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC): When in doubt, a concentrate is viewed as dynamic against the two fungi and bacteria if the inhibition zone was more noteworthy than 6 mm (22). Fifteen concentrations for the solutions were prepared (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75 μg/ml), these solution including nano-silver, partial purified lovastatin, immobilized lovastatin, and distilled water as control. Petri-dish plates consist of MuellerHinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with old culture (7×10 6 cells/ml) of the microbial strains, including Gram-positive: S.aureus, Gram-negative: E.coli and the yeast: C.albicans. Three wells (5 mm diameter) were performed and 0.1 ml of different concentration solutions were applied in each well. The results were observed and measuring the diameter of inhibition zones (clear zone without growth around the colony) (10). The determination of MBC and MFC was performed by sub-culturing portions of the agar from plates that showed no growth in the tests for determination of MICs. These agar were transferred respectively into plates containing nutrient agar for bacteria and Sabouraud dextrose agar for Candida albicans, then incubated at 37 and 28 °C for bacteria and yeast, respectively, for 24 hours and were observed for growth. If there was no growth, the solutions were identified as bactericidal or fungicidal (24). The cytotoxic effect of partial purified lovastatin: The strategy of this experiment (in vitro) was performed to examine the conceivable cytotoxic impact of standard and partial purified lovastatin on tumor cell lines (Michigan cancer foundation-7, MCF-7) and normal cell line WRL 68 (human liver cell line). The cytotoxic effect study was according to the manufacturer's instructions (27): The cells (1 x 10 4 to 1 x 10 6 cells/ml) were cultured in 96-well plates to end volume of 200 μl complete culture medium for each well. The plates were wrapped with a sterile parafilm, gently stirred and incubated for 24 hours at 37 ° C with 5% CO2. After the incubation, remove of the medium, and 200 μl of a 2fold various dilution of the standard and partial purified lovastatin (6.25, 12.5, 25, 50, 100, 200, 400 μg/ml) was added to the wells. Triplicate was performed at each concentration and control. Then incubation of the plates for 48 hours at 37°C with 5% CO2. Then, 10 μl of MTT solution was added to each well. The plates were incubated for 4 hours with 5 % CO2 at 37 °C. The medium was then neatly segregated, and in each well 100 μl of DMSO solution was added then incubated for 5 minutes. Absorbance was measured using an ELISA reader at a wavelength of 575 nm. Statistical analysis was performed on the optical density readings to calculate the IC50. According to the following equation: Viability (%) = (Optical density of sample / Optical density of control (live cells)) × 100


INTRODUCTION
Lovastatin is polyketide components, created or produce via certain fungi during their secondary metabolism. Lovastatin, also known as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors, are a class of lipid-lowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. The statins act as competitive inhibitors for HMG-CoA reductase, a rate constraining advance of cholesterol biosynthesis (2). Statins hinder the change of HMG-CoA to mevalonic acid in the mevalonate pathway (18). Production of lovastatin by submerged fermentation and solid state fermentation has been widely investigated and commonly, filamentous fungi exhibit tremendous potentiality (7). Aspergillus terreus is a filamentous ascomycota, which is significant provenance for generation of lovastatin (6). The immobilization methods are used for the binding of cells, organelles, compounds, proteins (3), or different materials onto a strong help, into a strong lattice or held by a membrane, so as to build their stability and make conceivable their rehashed or proceeded with employ (16). Silver particles having fine or ultrafine sizes have attracted scientific interest because of their unusual properties compared to bulk metal (19). Colloidal particles because of their quantum size effects and surface effects reveal excellent electrical conductivity, catalytical activity, chemical stability and antimicrobial activity (14). Interestingly, some studies have demonstrated an antimicrobial potential for statins against different bacterial species. For example, simvastatin was able to inhibit host-cell invasion and Staphylococcus aureus growth. In addition, lovastatin, atorvastatin, simvastatin and rosuvastatin showed activity against several reference bacteria, yeasts and clinical isolates (11). Lovastatin and associated components indicated wonder impacts on tumor cells however site of activity and mechanism of activity is ineffectively comprehended. Concentrate done via Xiangli et.al. uncovered anti-proliferative impacts of lovastatin on malignant cells. The experiment was performed on the tumor in human glioblastoma cells and decrease in the malignancy was showed by lovastatin through inactivation of RAS farsonylation (29). The purpose of this study was to immobilization of lovastatin produced from local isolate Aspergillus terreus A50 using solid state fermentation with nano-silver and used of lovastatin in various applications.

Lovastatin production
Collection of seventy three local fungal isolates from different areas, and screening for lovastatin production. The local isolate Aspergillus terreus A50 was best isolate for lovastatin production through submerged fermentation. The optimum conditions for lovastatin production by local isolate A.terreus A50 were used solid state fermentation (SSF) with media contain wheat bran and oat bran (1:1 w:w), sodium acetate, moisture ratio (1.2:1 v:w), pH (7), incubation temperature 30 °C and incubation period (6 days). Local isolate of A.terreus A50 was cultured on potato dextrose agar. Then lovastatin production from this isolate were performed by using a medium mention above and the growth was elicited with 1 ml (1x10 6 cells/ml)/5gm media of S.cerevisiae after 48 hours of culture.

Extraction of lovastatin
After the end of SSF operation, the concentration of lovastatin was measured. First, the culture was extracted in 250 ml Erlenmeyer's flask with ethyl acetate (pH 3.0). The mixtures was then incubated in rotating shaker with 140 rpm at 28 ºC for 2 h. Next, filtration was done using Whatman filter paper (No. 1) for separation of the biomass from the filtrate. Then stored the supernatant in glass bottles at 4 ºC until use for additional examination as crude extraction (26).

Estimation and purification of lovastatin
Lovastatin was estimated based on the method described by Mielcarek et al. (21) as follows: One ml of trifluroacetic acid (1%) was mixed with one ml of the supernatant and incubated for 10 min. A 0.5 ml from these mixture was diluted 10 times with methanol and its absorb-ance was estimated at 238 nm using UV-Visible Spectrophotometer. The lovastatin concentration in the sample was determined by plotting the O.D values on standard chart. Three ml of mixture from 0.5 ml trifluroacetic acid (1%) and 2.5 ml methanol were used as control. The purification of lovastatin produced by local isolate A.terreus A50 was performed by silica gel column chromatography (using silica gel column (67-1.5 cm) (60-120 Mesh) eluted with chloromethane :ethyl acetate (70:30 v:v), 3 ml for each fraction with flow rate 20 ml/ hour).

Effects of partial purified lovastatin on HMG-CoA Reductase activity
The study of the partial purified lovastatin effect on HMG-CoA reductase activity was performed by using kit (colorimetric) from abcam company. This experiment was achieved using 10 mM of standard, partial purified lovastatin, and atorvastatin (present in kit as standard inhibitor) . The enzyme was separately incubated with each inhibitor at 37 ºC each 2 min for 10 min. The the percentage (%) of enzyme inhibition was estimated according to the producer of kit (colorimetric) from abcam company. The enzyme inhibition was estimated according to the equation: ) }X 100. A= Absorbance, T= Time Thermal stability of lovastatin for HMG-CoA reductase inhibition: A 100 µl of standard and partial purified lovastatin separately were incubated in water bath at different temperature degrees (20,25,30,35,40,45,50, 55, and 60) ᴼC for 30 min, then the test tubes containing the lovastatin were transferred directly to cold water. The impacts of inhibitors on HMG-CoA Reductase activity were determined by using 5 µl (10 mM) of standard and partial purified lovastatin. The enzyme was separately incubated with inhibitors at 37 ºC each 2 min for 10 min. The enzyme inhibition % was estimated according to the same previous equation. Effect of different storage time of partial purified lovastatin on HMG-CoA reductase inhibition: A 100 µl of standard and partial purified lovastatin separately were incubated in water bath at 40 ᴼC for different time 2, 4, 6, 8, 10, 24, 48 hours. The test tubes containing the lovastatin were then transferred directly to cold water bath. The effects of inhibitors on HMG-CoA Reductase activity were investigated by using 5 µl (10 mM) of standard and partial purified lovastatin. The enzyme was separately incubated with inhibitors at 37 ºC each 2 min for 10 min. The enzyme inhibition % was estimated according to the same previous equation. Immobilization of lovastatin (Binding Nanosilver with lovastatin): Lovastatin was suspended in deionizing water and sonicated for 15 min. Next, 2 ml of lovastatin suspension (0.5 mg/ml) was transferred to tube and mixed with 2 ml of standard silver nanoparticles of concentration 0.7 mg/ml. After incubation for 24 hours the solutions were centrifuged 10000 rpm, for 10 minutes at room temperature, the supernatant was transferred to tube and ignores. While the precipitate was washed for four times, then were subjected to complete analysis via ultraviolet-visible spectrophotometry and ATR-FTIR analysis, for detect the binding between lovastatin and standard silver nanoparticles (28).

Lovastatin applications Antimicrobial activity of lovastatin:
The bacterial isolates were obtained from department of Biotechnology/College of Science/ University of Baghdad, bacteria were: Grampositive: Staphylococcus aureus and Enterococcus faecalis, Gram-negative: Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa and the yeast: Candida albicans. The bacteria and yeast were sub-cultured on nutrient broth and incubated at 37°C, while yeast in 28 ºC for 48 hours and then kept in the refrigerator for next experiments. The various concentrations of partial purified lovastatin were prepared using distilled water as solvent, concentration included 10, 50, and 75 μg/ml. The antibiotic (metronidazole) was prepared via distilled water with 50 μg/ml concentration. The metronidazole was used as control (1). Determination of lovastatin activity by agar diffusion method: According to Obeidat et al. (23), petri-dish plates contained of Mueller-Hinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with an overnight culture of indicators strains. Five wells (5 mm diameter) were made into the (two plates) agar via corkborer and three wells were loading with 0.1 ml of the partial purified lovastatin with different concentration (10, 50, and 75 μg/ml) and standard lovastatin with concentration of 75 μg/ml, as well as the metronidazole and distilled water were used as control. The inoculums volume was balanced in order to convey last inoculums of around 7×10 6 cells/ml, cells enumeration was doing by a hemocytometer. Incubation of bacteria were performed at 37 ºC and of yeast at 28 ºC for 24 hours. The assessment of antibacterial and antifungal activity was based on diameter measurement of the inhibition zone formed around the well. Antimicrobial activity of immobilized lovastatin: Petri-dish plates of Mueller-Hinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with old culture (7×10 6 cells/ml) of same microbial strains that mention above. Five wells (5 mm diameter) were made into (two plates) the agar via cork-borer and 0.1 ml of different solutions were applied in each well, these solution including the 75 μg/ml of standard lovastatin, partial purified lovastatin, immobilized lovastatin, nano-silver, and distilled water as control. The assessment of antibacterial and antifungal activity was based on diameter measurement of the inhibition zone formed around the well (23). Determination of minimum inhibition concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC): When in doubt, a concentrate is viewed as dynamic against the two fungi and bacteria if the inhibition zone was more noteworthy than 6 mm (22). Fifteen concentrations for the solutions were prepared (5,10,15,20,25,30,35,40,45, 50, 55, 60, 65, 70, and 75 μg/ml), these solution including nano-silver, partial purified lovastatin, immobilized lovastatin, and distilled water as control. Petri-dish plates consist of Mueller-Hinton agar for bacteria and Sabouraud dextrose agar for C.albicans were used. Agar media were cultured with old culture (7×10 6 cells/ml) of the microbial strains, including Gram-positive: S.aureus, Gram-negative: E.coli and the yeast: C.albicans. Three wells (5 mm diameter) were performed and 0.1 ml of different concentration solutions were applied in each well. The results were observed and measuring the diameter of inhibition zones (clear zone without growth around the colony) (10). The determination of MBC and MFC was performed by sub-culturing portions of the agar from plates that showed no growth in the tests for determination of MICs. These agar were transferred respectively into plates containing nutrient agar for bacteria and Sabouraud dextrose agar for Candida albicans, then incubated at 37 and 28 °C for bacteria and yeast, respectively, for 24 hours and were observed for growth. If there was no growth, the solutions were identified as bactericidal or fungicidal (24). The cytotoxic effect of partial purified lovastatin: The strategy of this experiment (in vitro) was performed to examine the conceivable cytotoxic impact of standard and partial purified lovastatin on tumor cell lines (Michigan cancer foundation-7, MCF-7) and normal cell line WRL 68 (human liver cell line). The cytotoxic effect study was according to the manufacturer's instructions (27): The cells (1 x 10 4 to 1 x 10 6 cells/ml) were cultured in 96-well plates to end volume of 200 µl complete culture medium for each well. The plates were wrapped with a sterile parafilm, gently stirred and incubated for 24 hours at 37 ° C with 5% CO 2 . After the incubation, remove of the medium, and 200 µl of a 2-fold various dilution of the standard and partial purified lovastatin (6.25, 12.5, 25, 50, 100, 200, 400 µg/ml) was added to the wells. Triplicate was performed at each concentration and control. Then incubation of the plates for 48 hours at 37°C with 5% CO 2 . Then, 10 µl of MTT solution was added to each well. The plates were incubated for 4 hours with 5 % CO 2 at 37 °C. The medium was then neatly segregated, and in each well 100 µl of DMSO solution was added then incubated for 5 minutes. Absorbance was measured using an ELISA reader at a wavelength of 575 nm. Statistical analysis was performed on the optical density readings to calculate the IC50. According to the following equation: Viability (%) = (Optical density of sample / Optical density of control (live cells)) × 100 %.

RESULTS AND DISCUSSION
Impact of lovastatin on HMG-CoA Reductase activity: The effects of inhibitors on HMG-CoA Reductase activity were determined by using 10 mM of standard and partial purified lovastatin. The results of this experiment show that HMG-CoA Reductase activity was inhibited at 10 mM of standard and partial purified lovastatin with specific activity 0.056 and 0.062 U/mg protein respectively, compared with control ( Figure 1), while (10 mM) atorvastatin (in kit as standard inhibitor) inhibit the HMG-CoA Reductase activity with specific activity 0.039 U/mg protein.

Figure 1. Effect of standard and partial purified lovastatin on HMG-CoA reductase activity
Peter and Jones (25), found that the statins have proved to be potent therapies for reducing elevated low-density lipoprotein (LDL) cholesterol and lessening the risk of coronary heart disease (CHD) and related events via inhibiting HMG-CoA Reductase enzyme. Zipp,et.al. (30), proved that brain cholesterol levels in mice were reduced after treatment with 100 mg/kg of lovastatin, no reduction was observed in guinea pigs treated with much higher doses of simvastatin over the same period of time, duo to the lovastatin is more effect on of HMG-CoA Reductase enzyme than simvastatin.

Thermal stability of lovastatin for inhibition of HMG-CoA reductase activity:
The results of this study demonstrated that the standard and partial purified lovastatin were stable in temperatures between 20-40 ᵒC, then the stability begun to decrease at 45 ᵒC ( Figure 2). This decrease in lovastatin may be due to the thermal effect on the lovastatin activity and/or structure.

Figure 2. Effect of different temperature on inhibitors (standard and partial purified lovastatin) for of HMG-CoA reductase inhibition
The literature mention that the storage of lovastatin must be at controlled room temperature 20-25 °C, and must avoid excessive heat and humidity. Ho et.al. (13), demonstrated that lovastatin exhibits higher thermal stability and lower degradation rate than simvastatin. Can-dyrine et.al. (9), showed that the best temperature for lovastatin storage was from 22 to 25 ᵒC.

Lovastatin stability at different storage time
The results of this study showed that the standard and partial purified lovastatin were stable in storage time between 1-8 hours, then the stability begun to decrease after the ten hours ( Figure 3).This decreases in inhibition by lovastatin may be due to the thermal effect on the lovastatin structure. Some literature re-ported that the temperature of lovastatin storage was at 20-25°C, also lovastatin must be protected from light and stored in a wellclosed, light-resistant container (9).

Figure 3. Effect of different storage time on lovastatin for HMG-CoA reductase inhibition Immobilized lovastatin (Nanosilverlovastatin):
The results showed that the nanosilver was bound with partial purified lovastatin to form immobilized lovastatin. Whereas the maximum absorbance of free standard nano-silver, free partial purified lovastatin, and standard nano-silver plus partial purified lovastatin without binding after scanning at  ) and compared with ATR-FTIR analysis of partial purified lovastatin, the FTIR spectra of immobilized lovastatin shows that the peaks at 1616.24 cm −1 for lovastatin-silver nanoparticles is due to alkenes C=C group, that has been shifted to a higher wavenumber in comparison to the primary aromatic C=C of partial purified lovastatin (1560.30 cm −1 ), which suggested that the role of primary aromatic C=C group of partial purified lovastatin in reduction of lovastatin-silver nanoparticles. The lactone ring bands and other bands do not show any shift in lovastatin-silver nanoparticles as compared to partial purified lovastatin. Hence it can be deduced that formation of lovastatinsilver nanoparticles and lactone ring of partial purified lovastatin preserves its integrity (figure 5 and 6).  Table (1). The isolates were sensitive to partial purified lovastatin. Staphylococcus aureus and S.typhi more sensitive to partial purified lovastatin than other Gram negative, Gram positive bacteria, and yeast, with inhibition zones of 24 mm and 23 mm respectively, at concentration 75 μg/ml. The inhibition zone of E. faecalis was 18 mm while the inhibition zones of each of E.coli, C.albicans, and P.aeruginosa were 22, 20 and 13 mm, respectively, at concentration 75 μg/ml. Also, the other concentrations (10, and 50 μg/ml) of partial purified lovastatin were showed low inhibition zones compare with 75 μg/ml concentration. Base on the results, it can be said that concentrations of partial purified lovastatin (10, 50, and 75 μg/ml) gives antimicrobial activity against both bacterial and fungi isolates (Table 1) according to Muhammad and Muhammad (22). less sensitive to statins contrasted with their relating standard strains. Statins are a class of pharmaceutical widely used to treat high serum cholesterol. In addition, statins have socalled pleiotropic impact, which include the reduction of inflammation, immunomodulation, and anti-microbial effects (12).

Antimicrobial activity of immobilized lovastatin:
The results in Table 2 show that the tested solutions were effective in inhibiting microbial growth of test microorganisms with different potency. Immobilized lovastatin was more effective against gram negative bacteria than gram positive bacteria and yeast. Immobilized lovastatin possess higher antimicrobial activity when compared with other com-pounds. The range of inhibition zones for immobilized lovastatin, partial purified lovastatin, Nano-silver were of 31-48, 12-23, 12-17 mm, respectively. The highest antibacterial activity was obtained from immobilized lovastatin against S. aureus (48 mm) and the lowest antibacterial activity was against E. faecalis (31 mm). The results demonstrated that the in vitro antimicrobial activity of immobilized lovastatin were more efficient compared to their respective free forms. In addition, developed a simple technique for the conjugation of lovastatin with nano-silver needs functionalization process. The interaction between lovastatin and nano-silver is likely to be mediated by the adsorption of the lovastatin molecules on the nanoparticle surfaces. Rogowska, et.al., (28), found that ampicillin from an unmodified counterpart was less effective against Klebsiella pneumoniae and S.aureus, and only for E.coli is a strong synergistic effect. A similar phenomenon has been observed for amoxicillin-resistant strain S.aureus. The concomitant use of silver nanoparticles and amoxicillin in this case, revealed antagonistic effect. It is likely that nanoparticles (Ag) prevent the absorption of a large amount of ampicillin. Nanoparticles bind to the surface of bacterial cells. Brown et.al., (8), has shown that ampicillin-resistant bacterial strains such as P.aeruginosa, Enterobacter aerogenes or S.aureus are vulnerable to silver nanoparticles used with this antibiotic. Its performance was even higher than nanoparticles made of silver alone.

Determination of Minimum Inhibition Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)
Fifteen concentrations for the solutions were prepared (5, 10, 15, 20, 25, 30, Table 5. Chi-Square Tests of effect of partial purified lovastatin on MCF-7 cells    Partial purified lovastatin displayed a dosedependent pattern of piecemeal cytotoxicity starting from the lower concentration to its more strong inhibition at 400µg/ml, 24.575 % inhibition of WRL 68 cells line, and 51.273 % inhibition of MCF-7 cells. The outcomes of cytotoxic impact of partial purified lovastatin indicated that remedying MCF-7 cells at concentrations extending from 6.25 to 400 µg/ml for 24 hours demonstrated an important mortality in cell viability via rising the concentration in a portion subordinate example that came to up to 51.27 % killing at 400 µg/ml with an IC 50 of 138.1µg/ml ( figure 7). In addition, the treatment of MCF-7 cells with standard lovastatin showed an influence by applying the similar concentration range with an identified IC 50 of 112 µg/mL ( figure 8). On the other hand, figure (9 and 10) showed that it is possible to determine the effect of an unknown concentration of standard and partial purified lovastatin by using regression equations according to the following equations: Y= 1.56+4.65E-3x »for partial purified lovastatin. Y= 1.56+5.55E-3x »for standard lovastatin