الفهرس 
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Abstract
Progesterone is C-21 steroid hormone involved in menstrual cycle and use in maintenance of pregnancy exhibit a wide variety of end organ effects; in males it is necessary in production of corticosteroids and androgens.
Progesterone have more than one derivative of low molecular weight approximately 314 Dalton. They are not naturally occur but can be transformed by plants cell, callus cultures and microorganisms.
This study aimed to production of progesterone hormone and its metabolites from phyto-microbial cultures, then immunized into rabbits to produce antiserum helped in production of diagnosis kit for progesterone.
Tomato protoplast isolated from leaves tissues has proven to be especially useful for plant development and biotransformation of progesterone. Protoplast viability was determined by light microscope and staining with non fluorescent dye. Methylene blue was selected as non fluorescent dye for measuring cell viability and total cell number in preparations of tomato protoplasts.
Preliminary experiment was performed for choosing the most successful plant tissue to from callus. Data show that explants taken from roots and stem of healthy tomato failed in grow on MS medium (supplied with 0.148 mg/L NAA and 1.25 mg/L BA), most explants taken from middle veins of upper leaves were survived weigh grew fast and give calli without plantlets. Benzyladenine (BA) level choose for suitable callus growth was 1.25 mg/L medium of level BA increased above levels mentioned generating plantlets from growing calli results show that when explants cultured on MS medium supplemented with 2.25 mg/L medium on other contrary ,the level of BA decreased than 1.25 mg/L medium.
The In Vitro biotransformation of progesterone was done after 10-15 days of incubation under protoplast and calli cultures of tomato in flask containing 100 ml of MS medium with 1 μg of 2, 4-dichlorophenoxy acetic acid per ml and incubation at 28˚C. from cell or callus suspension culture of tomato plants it was possible to isolate five biotransformation products (5αhydroxyprogesterone; 20αhydroxyprogesterone; 20α6βhydroxy-progester-one; 11α-hydroxyprogesterone; 11α, 6β-hydroxyprogesterone) and unknown metabolite. The concentration of each product was 37.5; 45.0; 48.33; 51.67; 50.83 mg/ml (protoplast) 44.17; 39.17; 52.5; 47.5; 54.17 mg/ml (callus) respectively of initial quantity of progesterone added. After prolonged In Vitro incubation (10-15 days) the percentage of the unknown metabolite started to decline slightly.
TLC detection showed five metabolic compounds of five chemo types compared with standard reference components. The compounds were differed in spot area and percentage from the total area of spots. The qualities finger prints of the five compounds are an evident that all microbial cultures biotransformation includes qualitative alterations, which seems to have a genetic block in producing the compounds.
The biotransformation metabolites were separated by HPLC and detected at 240 nm. The absorbance at 240 nm indicated that 6β-hydroxyprogesterone structure in the ring was undistributed for all analytes. from the analysis of steroid metabolites, in addition to 6β and 20α-hydroxyprogesterone, two previously uncharacterised hydroxylated progesterone have been observed in HPLC chromatogram. At acetonitric: methanol: water (25:25:50 V/V/V) as mobile phase a good separation of the five metabolites was achieved.
For A.niger culture in this experiment two ml of spore suspention were used to inoculate each flask. The inoculums and the transformation media were the same in each instance. After 7 days growth a methanolic solution of progesterone was charged to each flask to achieve a final concentration of 50 mg/ml and incubated at 25˚C. The transformations were assayed form whole broth incubated for 10 days following initial progesterone was charged by TLC and HPLC.
All these metabolites produced in spectrum of different colours with anisaldhyde reagent. TLC detection showed two metabolic compounds compared with standard reference compounds. The compounds were differed in the spot area and percentage from the total area of spots.
The methanol extract of progesterone transformation products was resolved by HPLC using acetonitrile: methanol: water (25:25:50 v/v/v) as a mobile phase. The purified progesterone transformation products were run under the same condition and their retention time were used to identify these compounds in the total extract. The results do not give a complete picture of the transformation pattern. These products were isolated and identified from a progesterone transformation by A.niger. Two major transformation product of progesterone from the majority of this culture were obtained 11α, 6β-hydroxyprogesterone and 11α-hydroxyprogesterone concentration 50.06 and 41.67 mg/ml respectively. A.niger first adapts to progesterone transforming these substrate to 11α-hydroxyprogesterone and11α, 6β-hydroxyprogesterone. Although quantitative measurements of progesterone transformation to dihydroxyprogesterone derivatives had been made in a relatively few instance, the qualitative assay results indicate that quite high over all can be obtained. Transformation of progesterone for the 11α-hydroxyprogesterone derivative had been obtained in 90 % yield and 11α6β-hydroxyprogesterone derivative.
For B.subtilus Starter inoculum (50 ml) of B.subtilus 6,10 UFC (24hours old) was used to inoculate the transformation media. The culture transferred to a fresh 500 ml TYE media in 2 L baffled flasks and were shaken at 65˚C until mid-log phase of growth (3-4 hours). An methanolic solution of progesterone was added to the flasks to achieve a final concentration of 50 mg/ml and was re-incubated for further 24 hours. The transformations were assayed from whole broth by TLC and HPLC. All these biotransformation metabolites produced a spectrum of different colours with anisaldehyde reagent. The methanol extract of progesterone transformation product resolved by HPLC using acetonitrile: methanol: water (25:25:50 v/v/v),as a mobile phase. The purified progesterone transformation products were run under the same condition and the retention times were used to identify these products. The isolates of B.subtilus was capable of site selective hydroxylation of progesterone producing rare microbial transformation products by concentrations 5α-hydroxyprogesterone(41,67) mg/ml,6β-hydroxyprogesterone(29.17) mg/ml and 20α-hydroxyprogesterone(45.83) mg/ml and the other metabolites unidentified.
Progesterone is a steroid hormone with a molecular weight about 314 Dalton. So it conjugated with bovine serum albumin (BSA) to the appropriate succinate derivatives to produce good immunogens injected into rabbits. By taking 6 mg antigen emulsified with 3 ml Freunds complete adjuvant then injected into rabbits intradermal then booster injections were injected with three weakly intervals using Freund’s complete adjuvant four days before fusion, an intravenous injection 5μg of antigen in 0.1 ml PBS without adjuvant was administered. Then blood was collected and centrifuged to take antiserum.
The IgG fraction was partially purified by selective preparation with saturated ammonium sulphate. Mixed 3.0 mg alkaline phosphtase (AP) with 1.0 mg of purified antibodies (Ab), in a total of 0.5 ml (1ml) phosphate buffer (0.01 M) containing (PBS), Glutaraldhyde was added after dialysis to final concentration 0.2%, then allowed the reaction to proceed two hours at room temperature, then glycerol was added to 50%(v/v) for stabilization of conjugate and stored at -20˚C.
The amount of specific antibodies to progesterone in serum was determined by direct ELISA using a microplate. The titre of progesterone and biotransformation antisera for 11α-hydroxyprogesterone, 5α-hydroxyprogesterone, 6α-hydroxyprogesterone and 20α-hydroxy-progesterone were 1/512; 1/512; 1/1024; 1/1024 respectively. ELISA was used for measurement of anti-progesterone activity. Metabolites extracted of B.subtilus, A.niger, cells and calli tomato cultures (dilution 1/10) reacted positively. It was found that B.subtilus produced highest amount of biotransformation and A.niger or B.subtilus gave higher absorption values compared with calli cultures. B.subtilus gave higher absorption of progesterone 90 ng/ml followed by A.niger 71ng/ml then calli tomato cultures 47ng/ml and cells tomato cultures 41 ng/ml.
On the other hand antiserum of 5α-hydroxyprogesterone was cross reacted with other antigen prepared for 6α-hydroxyprogesterone, 20α-hydroxyprogesterone, 11α-hydroxyprogesterone, 20α6β-hydroxypro-gesterone and 11α6β-hydroxyprogesterone as well as original progesterone with different degrees of serological reactions.
Radioimmunoassay (RIA) was used for measurement of antiserum of progesterone activity. Although RIA is more sensitive 2 to 4 fold than less subject to variation than indirect ELISA, ELISA was performable in most instances to avoid handling, strong and disposing of radioactive materials. RIA is particularly useful when pigmentation and for plant enzyme interfere with ELISA data.