APPENDIX Reagents. Appendix. Alsever s solution Citric acid 0.55g Sodium citrate 8.0g D-glucose 20.5g Sodium Chloride 4.2g

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1 APPENDIX Reagents Alsever s solution Citric acid 0.55g Sodium citrate 8.0g D-glucose 20.5g Sodium Chloride 4.2g 1000 ml Barritt s reagent A α-naphthol 5.0g ethanol 95.0 ml α-naphthol was dissolved in ethanol with constant stirring. Barritt s reagent B Potassium hydroxide 40.0g Creatine 0.3g ml Potassium hydroxide was dissolved in 75 ml distilled water. After cooling the solution to room temperature, creatine was added and dissolved by stirring. The remaining 25 ml distilled water was added. Kovac s reagent p- dimethylaminobenzaldehyde 2.0g Isoamyl alcohol 30 ml Concentrated hydrochloric acid 10 ml p- dimethylaminobenzaldehyde was dissolved in isoamyl alcohol. Concentrated hydrochloric acid was added slowly and stored in a brown bottle. Methyl red solution Methyl red 0.1g Ethyl alcohol ml ml Methyl red was dissolved in ethyl alcohol and then the final volume was made upto 500 ml distilled water. i

2 α-naphthylamine reagent α-naphthylamine 0.1 g glacial acetic acid 5.7 ml 14.3 ml α-naphthylamine was dissolved in acetic acid and then distilled water was added to make the final volume upto 20 ml. Media composition Czapek dox s Broth and Agar* Sucrose 30g Sodiun Nitrate 2g K 2 HPO 4 1g Magnesium sulphate 0.5g Potassium chloride 0.5g Ferrous sulphate 0.01g Glucose minimal Agar A Potassium Dihydrogen phosphate 3g Disodium hydrogen phosphate 6g Ammonium chloride 2g 800ml Agar 20g B Glucose 8g MgSO 4.7H 2 O 0.1g Thiamine 1mg 200mg Autoclaving A and B separately and mix before pouring. ii

3 Malt Extract broth and Agar* 5 g of malt extract in 1000 ml of distilled or deionized water water and autoclaved at 121⁰ C for 20 min; ph 5.5. Mueller Hinton Broth and Agar Suspended 38g in 1000 ml distilled water and autoclaved at 121⁰ C for 20 min. Minimal glucose agar medium Solution A: Minimal medium: MgSO 4.7H 2 O 0.2g Citric Acid 2.0g K 2 HPO 4 (NH 4 ) 2 HPO g NaOH 0.66g Solution B: 20% Glucose solution (20 g of glucose in 100 ml) Solution C: 20 g agar in 700 ml of distilled water The solution A, B and C were separately prepared and autoclaved at 121⁰ C for 20 min. The solution A, B and C were mixed and poured to petridishes. Potato dextrose Broth and Agar* Potato 200g Dextrose 20g ph ml of distilled or deionized water and autoclaved at 121⁰ C for 20 min. Sabouraud s Broth and Agar* Dextrose 40g ph 6.2 iii

4 Simmon s citrate agar medium Sodium chloride 5.0 g Potassium dihydrogen phosphate 1.0g Ammonium dihydrogen phosphate 1.0g Magnesium sulphate 0.2g Sodium citrate 2.0g Bromothymol blue (w/v) 4 ml Agar 15g 1000 ml ph Top Agar Sodium Chloride 5.0g Agar 6.5g 1000 ml 6 gm agar in 100 ml distilled water and 5 ml were dispersed in test tube and then these test tubes were autoclaved at 121⁰ C for 20 min. Tryptone Soya Broth and Agar* Casein 15.0g Soya peptone 5.0g Sodium chloride 5.0g Yeast extract glucose Broth and Agar* Glucose 5g Yeast extract 3g ph 5.5 iv

5 Yeast peptone Dextrose Broth Dextrose Starch Yeast extract 4g ph 5.5 Yeast Malt Broth and Agar* 5.0g Yeast Extract 3.0g Malt Extract 3.0g Dextrose 10.0g ph 6.2g Yeast extract glucose broth and Agar* Glucose 5g Yeast extract 3g ph 5.5 Buffer preparation Phosphate buffer solution (PBS) Sodium Chloride 9.0g Potassium dihydrogen phosphate 0.21g Disodium hydrogen phosphate anhydrous 0.726g ph 7.2 Mix all the ingredients in small amount of water then make up the volume to 1L v

6 Appl Biochem Biotechnol (2014) 173: DOI /s y Isolation, Purification, and Characterization of Antimicrobial Compound 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3- (2-methoxy-phenyl)-chromen-4-one from Penicillium sp. HT-28 Harpreet Kaur & Daljit Singh Arora & Vishal Sharma Received: 11 December 2013 / Accepted: 19 May 2014 / Published online: 7 June 2014 # Springer Science+Business Media New York 2014 Abstract A fungal culture (Penicillium sp., HT-28), isolated from soil has been evaluated for its bioactivity, which showed broad spectrum antimicrobial activity and was effective against methicillin-resistant Staphylococcus aureus (MRSA) also. Statistical optimization of the medium by response surface methodology (RSM) enhanced the antimicrobial activity up to 1.8-fold. Column chromatography was used to isolate the active compound (A), which was characterized to be 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one by various spectroscopic techniques such as infrared (IR), 1 H and 13 C nuclear magnetic resonance (NMR) spectra, and mass spectroscopy. Minimum inhibitory concentration (MIC) of the active compound (A) ranged from 0.5 to 15μg/mL. Viable cell count studies of the active compound (A) showed S. aureus, Escherichia coli, Staphylococcus epidermidis, and Salmonella typhimurium 1 to be the most sensitive. The compound retained its bioactivity after treating it at 100 C for 1 h. Furthermore, the compound (A) when tested for its biosafety was found neither to be cytotoxic nor mutagenic. The study demonstrated that an apparently novel compound isolated from Penicillium sp. (HT-28) seems to be a stable and potent antimicrobial. Keywords Antimicrobial. Fungi. Isolation. Penicillium. Purification. Resistance Introduction Natural products are of great importance for their use in different fields for the welfare of mankind. The unexplored products from different natural resources are gaining importance for their use in various industrial applications. Fungi produce a variety of secondary metabolites which can be used as antimicrobials, antihyperlipidimics, and immunosuppressants [1]. Emergence and spread of antimicrobial resistance is a major H. Kaur: D. S. Arora (*) Microbial Technology Laboratory, Department of Microbiology, Guru Nanak Dev University, Amritsar , India daljit_02@yahoo.co.in V. Sharma Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar , India

7 Daljit Singh, et al. Int J Pharm 2014; 4(1): ISSN International Journal of Pharmacy Journal Homepage: Research Article CODEN: IJPNL6 PRODUCTION, OPTIMIZATION AND CHARACTERIZATION OF ANTIMICROBIAL COMPOUND FROM ASPERGILLUS SP Daljit Singh Arora a *, Harpreet kaur a, Jemimah Gesare Onsare a and Vishal Sharma b a Microbial Technology Laboratory, Department of Microbiology, Guru Nanak Dev University, Amritsar , India b Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar , India. *Corresponding author daljit_02@yahoo.co.in ABSTRACT Fungi have been reported to be active producers of secondary metabolites. In this study, a fungal isolate (Aspergillus sp) isolated from soil has been evaluated for its antimicrobial activity. The activity was studied under various physio-chemical parameters, such as ph, temperature, incubation period, carbon and nitrogen sources. The best antimicrobial activity was observed in the production medium having ph 5-7, on fifth day of incubation at 25 ºC when grown as static culture. Starch was the most promising carbon source, while yeast extract and soyabean meal acted as best nitrogen sources. Butanolic extract was comparable to standard antibiotics in contrast to aqueous extract. Response surface analysis showed that the antimicrobial activity was enhanced by 1.25 folds in S.aureus, 1.28 folds (S.epidermidis), 1.6 folds (K.pneumoniae 1), 1.37 folds (C.albicans), 1.38 folds (MRSA). Characterization of the purified compound responsible for antimicrobial activity was carried out by various analytical procedures i.e. TLC, HPLC, NMR and IR. MIC of the butanolic extract ranged from (0.016mg/ml-18mg/ml) while purified compound exhibited lower MIC value of 6µg/ml, 20 µg/ml and 20 µg/ml respectively for S.epidermidis, C.albicans and MRSA. VCC (Viable cell count) studies revealed E.coli to be the most sensitive and demonstrated 100% killing at 0 hr. Butanolic extract (crude) and the purified compound were found to be neither cytotoxic nor mutagenic. Key words: Aspergillus, Antimicrobial, Minimum inhibitory concentration, Viable cell count, Bioactive compound, Resistance. INTRODUCTION Microbial populations are the most promising sources of natural secondary metabolites. Fungi produce a wide range of such products with therapeutic value as antibiotics, along with many other bioactive compounds of pharmaceutical and agricultural use. Many Aspergilli have been reported to be the active producers of different secondary metabolites with various bioactivities such as antiviral, antimicrobial, tumor suppressing, antihypercholesterolemic and immunosuppressant activities [1]. Soil holds an enormous biodiversity that can be screened for various bioactive metabolites. Number of fungi showing different biological activities have been listed in the literature, still a lot remains untapped from diverse soil habitats. Further, bacterial resistance is spreading throughout the world, revealing the steadily decreasing potencies of prevalent antibiotics [2]. Strains of methicllin resistant Staphylococcus aureus have increased to an alarming number leading to many infectious diseases, so there is a need to explore and expand the spectrum of suitable organisms yielding novel antimicrobials. Keeping this in mind, the present study was designed to screen the fungi from soil collected from different areas of Punjab (30 4 N 75 5 E) for their antimicrobial activity. One such promising isolate, was perused further for optimization of various physiochemical parameters to enhance the production of antimicrobial agent/s. Minimum inhibitory concentration and viable cell count has been carried out to check the bacteriostatic or bactericidal nature of the butanolic extract of Aspergillus sp. Purification 157