Determination of hepatitis C virus subtype prevalent in Sindh, Pakistan: a phylogenetic analysis.

Hepatitis is a major public health issue, affecting 10-17 million people worldwide, with its prevalence continuously increasing. The Hepatitis C virus (HCV) is responsible for liver related diseases, which include liver cirrhosis, hepatocellular carcinoma, and chronic hepatitis. Pakistan is experiencing a serious rise in HCV cases. This study aimed to assess the prevalence and distribution of HCV genotypes in Sindh, Pakistan. Serum samples from HCV-positive patients were collected from various local hospitals in Sindh. These samples were first screened for HCV antibodies using ELISA. Samples that tested positive for HCV RNA underwent further genotyping through sequencing using the standard Sanger method. The genotypes were identified by comparing the sequences with those available in the National Center for Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed. The phylogenetic analysis showed that all isolates in this study were clustered with genotypes 3a and 3b, except for one sequence that was clustered with genotype 1a. No isolates were found to be clustered with reference genomes of genotypes 2, 4, 5, 6, and 7 suggesting that genotype 3a is endemic in this region. The analyzed sequences demonstrated a 98% similarity with reference and isolated sequences. In summary, sequencing of the HCV 5' UTR essential for identifying the predominant genotype of HCV RNA in the Sindh region Further research on the distribution of HCV genotypes in other regions of Pakistan could aid in improving screening processes, identifying more effective treatment options, and developing suitable prevention strategies.

Plant anthraquinones: Classification, distribution, biosynthesis, and regulation.

Anthraquinones are polycyclic compounds with an unsaturated diketone structure (quinoid moiety). As important secondary metabolites of plants, anthraquinones play an important role in the response of many biological processes and environmental factors. Anthraquinones are common in the human diet and have a variety of biological activities including anticancer, antibacterial, and antioxidant activities that reduce disease risk. The biological activity of anthraquinones depends on the substitution pattern of their hydroxyl groups on the anthraquinone ring structure. However, there is still a lack of systematic summary on the distribution, classification, and biosynthesis of plant anthraquinones. Therefore, this paper systematically reviews the research progress of the distribution, classification, biosynthesis, and regulation of plant anthraquinones. Additionally, we discuss future opportunities in anthraquinone research, including biotechnology, therapeutic products, and dietary anthraquinones.

Study of drug resistance-associated genetic mutations, and phylo-genetic analysis of HCV in the Province of Sindh, Pakistan.

Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. The aim of the current study was to identify such mutations in the NS3, and NS5B genes in DAAs treatment-naïve Pakistani chronic HCV 3a patients. Peripheral blood samples were collected from 233 chronic HCV 3a patients at different tertiary care hospitals in Karachi, Pakistan, between August 2020 to September 2021. PCR-amplified target regions of the NS3/NS5B gene were subjected to Sanger sequencing to identify resistance-associated mutations. Phylogenetic analysis of the identified amino acid sequences was performed using HCV3a sequences of the global population in the virus pathogen resource (VIPR) database. Sequence analysis identified five amino acid mutations, Leu36Pro, Gln41His, Gln80Lys/Arg, Ala156Tyr, and Gln168Arg in the NS3 region, and two mutations Leu159Phe and Cys316Arg in the NS5B region. Phylogenetic analysis revealed a high genetic diversity in the studied isolates. Overall, the prevalence of resistance-associated substitutions was almost similar to other geographic regions worldwide. This data could be helpful in selecting the most effective treatment regimen for HCV chronically infected people in Pakistan.

Identification of Non-steroidal Aromatase Inhibitors via In silico and In vitro Studies

INTRODUCTION: Breast cancer is the most common cancer affecting women worldwide, including Pakistan. More than half of breast cancer patients have hormone-dependent breast cancer, which is developed due to the over-production of estrogen (the main hormone in breast cancer). METHOD: The biosynthesis of estrogen is catalyzed by the aromatase enzyme, which thus serves as a target for the treatment of breast cancer. During the current study, biochemical, computational, and STD-NMR methods were employed to identify new aromatase inhibitors. A series of phenyl-3- butene-2-one derivatives 1-9 were synthesized and evaluated for human placental aromatase inhibitory activity. Among them, four compounds 2, 3, 4, and 8 showed a moderate to weak inhibitory activity (IC50 = 22.6 - 47.9 µM), as compared to standard aromatase inhibitory drugs, letrozole (IC50 = 0.0147 ± 1.45 µM), anastrozole (IC50 = 0.0094 ± 0.91 µM), and exemestane (IC50 = 0.2 ± 0.032 µM). Kinetic studies on two moderate inhibitors, 4 and 8, revealed a competitive- and mixed-type of inhibition, respectively. RESULT: Docking studies on all active compounds indicated their binding adjacent to the heme group and interaction with Met374, a critical residue of aromatase. STD-NMR further highlighted the interactions of these ligands with the aromatase enzyme. CONCLUSION: STD-NMR-based epitope mapping indicated close proximity of the alkyl chain followed by an aromatic ring with the receptor (aromatase). These compounds were also found to be non-cytotoxic against human fibroblast cells (BJ cells). Thus, the current study has identified new aromatase inhibitors (compounds 4, and 8) for further pre-clinical and clinical research.

Metabolomics analysis reveals the accumulation patterns of flavonoids and phenolic acids in quinoa (Chenopodium quinoa Willd.) grains of different colors.

Quinoa grains are gaining increasing popularity owing to their high nutritional merits. However, only limited information is available on the metabolic profiles of quinoa grains. In this study, we determined the metabolic profiles of black, red, and white quinoa grains via an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)-based metabolomics. A total of 689 metabolites were identified, among which 251, 182, and 317 metabolites displayed different accumulation patterns in the three comparison groups (Black vs Red, Black vs White, and Red vs White), respectively. In particular, flavonoid and phenolic acid contents displayed considerable differences, with 22 flavonoids, 5 phenolic acids, and 1 betacyanin being differentially accumulated among the three quinoa cultivars. Additionally, correlation analysis showed that flavonoids and phenolic acids could act as betanin co-pigments in quinoa grains. In conclusion, this study provides comprehensive insights into the adequate utilization and development of novel quinoa-based functional foods.

Genome-wide identification of key enzyme-encoding genes and the catalytic roles of two 2-oxoglutarate-dependent dioxygenase involved in flavonoid biosynthesis in Cannabis sativa L.

BACKGROUND: Flavonoids are necessary for plant growth and resistance to adversity and stress. They are also an essential nutrient for human diet and health. Among the metabolites produced in Cannabis sativa (C. sativa), phytocannabinoids have undergone extensive research on their structures, biosynthesis, and biological activities. Besides the phytocannabinoids, C. sativa is also rich in terpenes, alkaloids, and flavonoids, although little research has been conducted in this area. RESULTS: In this study, we identified 11 classes of key enzyme-encoding genes, including 56 members involved in the flavonoid biosynthesis in C. sativa, from their physical characteristics to their expression patterns. We screened the potentially step-by-step enzymes catalyzing the precursor phenylalanine to the end flavonoids using a conjoin analysis of gene expression with metabolomics from different tissues and chemovars. Flavonol synthase (FLS), belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, catalyzes the dihydroflavonols to flavonols. In vitro recombinant protein activity analysis revealed that CsFLS2 and CsFLS3 had a dual function in converting naringenin (Nar) to dihydrokaempferol (DHK), as well as dihydroflavonols to flavonols with different substrate preferences. Meanwhile, we found that CsFLS2 produced apigenin (Api) in addition to DHK and kaempferol when Nar was used as the substrate, indicating that CsFLS2 has an evolutionary relationship with Cannabis flavone synthase I. CONCLUSIONS: Our study identified key enzyme-encoding genes involved in the biosynthesis of flavonoids in C. sativa and highlighted the key CsFLS genes that generate flavonols and their diversified functions in C. sativa flavonoid production. This study paves the way for reconstructing the entire pathway for C. sativa's flavonols and cannflavins production in heterologous systems or plant culture, and provides a theoretical foundation for discovering new cannabis-specific flavonoids.

Characterization and expression analysis of MATEs in Cannabis sativa L. reveals genes involving in cannabinoid synthesis.

The medicinal plant Cannabis sativa L. (C. sativa) accumulates plant cytotoxic but medicinally important cannabinoids in glandular trichomes and flowers of female plants. Although the major biosynthetic pathway of cannabinoids has been revealed, their transportation mechanism is still unknown. Multidrug and toxic compound extrusion proteins (MATEs) can transport plant metabolites, ions and phytohormones intra and inter-cellularly. MATEs could have the potential to translocate cannabinoids or their synthetic intermediates to cellular compartment, thus protecting them from unwanted modifications and cytotoxicity. In this study, we performed a genome-wide identification and expression analysis of Cannabis sativa MATEs (CsMATEs) and revealed 42 CsMATEs that were classified phylogenetically into four conserved subfamilies. Forty-two CsMATEs were unevenly distributed on 10 chromosomes, with 50% CsMATEs were physically adjacent to at least one another CsMATEs and 83% CsMATEs localized on plasma membrane. Tandem duplication is the major evolutionary driving force for CsMATEs expansion. Real-time quantitative PCR revealed CsMATE23, CsMATE28 and CsMATE34 mainly expressed in flower, whereas CsMATE17 and CsMATE27 showed strong transcription in root. Light responsive cis-acting element was most abundant in promoters of CsMATE23, CsMATE28 and CsMATE34. Finally, the contents of cannabinoids and corresponding biosynthetic intermediates as well as expressions of CsMATE28 and CsMATE34 were determined under UV-B treatment, among which strong correlation was found. Our results indicates that CsMATEs might involve in biosynthesis of cannabinoids and has the potential to be used in heterologous production of cannabinoids.

Identification of quinoline derivatives as growth inhibitors of MDR pathogen Klebsiella pneumoniae.

Aims: This study was aimed to identify compounds with significant inhibitory potential against multidrug-resistant (MDR), multidrug-sensitive and clinical isolates of Klebsiella pneumoniae. Materials & methods: Antibacterial activity of the nitroquinoline derivatives was assessed by micro-plate Alamar Blue assay. Results: Nitroquinoline derivatives 9, 11 and 14 showed inhibitory activity against MDR K. pneumoniae. Docking studies of these compounds with topoisomerase IV of K. pneumonia indicated the interactions of these compounds at the active site residues of enzyme near to cofactor (Mg+2). Furthermore, compound 11 was identified as a reactive oxygen species (ROS) inducer. None of the compounds showed hemolytic effect. Conclusion: This study was designed to identify compounds active against MDR K. pneumoniae which causes infections, such as pneumonia and urinary tract infections.

Insight into isolation and elucidation of cytotoxic ergostanoids from the mushroom Sarcosphaera crassa (Santi) Pouzar: An edible mushroom.

Sarcosphaera crassa is a mushroom consumed in Europe and Anatolia after being cooked well. The cytotoxic activity of the extracts of unbaked S. crassa against MCF7, HT29, HeLa cancer cell lines and toxicity against PDF fibroblast healthy cell lines were studied using MTT assay. Acetone and methanol extracts of the mushroom exhibited significant cytotoxic activity. Further investigation of cytotoxic extracts afforded two new fatty acid sterols (1-2), a new ergosterol glycoside (4), and seven known compounds, including a fatty acid sterol (3), a steroid glycoside (5), two ergostanoids (6-7) and three sugars (8-10). These compounds were identified as brassicasteryl heptadecanoate (1), brassicasteryl palmitoleate (2), brassicasteryl linoleate (3), brassicasterol ß-ᴅ-xylofuranoside (4), brassicasterol ß-ᴅ-glucoside (5), brassicasterol (6), ergosterol-endoperoxide (7), mannitol (8), erythritol (9) and turanose (10). Among them, 7 exhibited a moderate cytotoxic activity against HeLa (IC50: 70.1 ± 2.0 µg/mL) and high activity against HT29 (IC50: 38.8 ± 0.9 µg/mL), and MCF7 (IC50: 62.9 ± 1.3 µg/mL) cancer cell lines. Compounds 4, 5, and 6 also exhibited significant cytotoxic activity against HT29 and MCF7. Moreover, all compounds exhibited weak toxicity against PDF healthy cell lines. This study indicates the potential use of Sarcosphaera crassa as a natural source of cytotoxic ergostanoids, which can be considered a dietary supplement for cancer prevention.

A new steroidal alkaloid from Fritillaria michailovskyi Fomin.

A new steroidal alkaloid, michainine (1), was isolated from Fritillaria michailovskyi Fomin, along with nine known compounds 2-10 of different classes, including ribonucleoside, steroids, and fatty acids, which were isolated for the first time from this plant. Their structures were elucidated through extensive spectroscopic techniques, as well as by comparing the data in the literature. Furthermore, the dichloromethane fraction of F. michailovskyi showed a positive butyrylcholinesterase inhibitory activity, along with non-cytotoxicity against 3T3 cell line.

Synthesis of 1,2,3,triazole modified analogues of hydrochlorothiazide via click chemistry approach and in-vitro α-glucosidase enzyme inhibition studies.

The current study was aimed to discover potent inhibitors of α-glucosidase enzyme. A 25 membered library of new 1,2,3-triazole derivatives of hydrochlorothiazide (1) (HCTZ, a diuretic drug also being used for the treatment of high blood pressure) was synthesized through click chemistry approach. The structures of all derivatives 2-26 were deduced by MS, IR, 1H-NMR, and 13C-NMR spectroscopic techniques. All the compounds were found to be new. Compounds 1-26 were evaluated for α-glucosidase enzyme inhibition activity. Among them, 18 compounds showed potent inhibitory activity against α-glucosidase with IC50 values between 24 and 379 µM. α-Glucosidase inhibitor drug acarbose (IC50 = 875.75 ± 2.08 µM) was used as the standard. Kinetics studies of compounds 6, 9, 11, 12, 15, 20, 23, and 24 revealed that only compound 15 as a mixed-type of inhibitor, while others were non-competitive inhibitors of α-glucosidase enzyme. All the compounds were found to be non-cytotoxic when checked against mouse fibroblast 3T3 cell line.

Peptide conjugates of 18ß-glycyrrhetinic acid as potent inhibitors of α-glucosidase and AGEs-induced oxidation.

18ß-Glycyrrhetinic acid (18ß-GA) is known for several biological activities, and has been the focus of extensive research for the development of therapeutic agents. In the current study, 18ß-GA-peptide conjugates 2-11 were evaluated for their in vitro α-glucosidase inhibitory and antiglycation activities. Structure-activity relationship (SAR) established and molecular interactions of active bioconjugates with the enzyme's binding sites were predicted through molecular modeling approach. In tripeptide moiety of conjugates 2-11, peptide residue at position 1 was found to have a significant role on α-glucosidase inhibition. The most active 18ß-GA-peptide conjugates 5 (18ß-GA-Cys1-Tyr2-Gly3), and 8 (18ß-GA-Pro1-Tyr2-Gly3) exhibited several-fold potent α-glucosidase inhibition (IC50 values 20-28 µM), as compared to standard drug acarbose (IC50 = 875.8 ± 2.10 µM). Kinetic studies of potent compounds, 4-8 revealed that conjugate 5 exhibits competitive-type of inhibition, while conjugates 6-8 showed a non-competitive type of inhibition. The simulation studies also supported the kinetic results that conjugate 5 (18ß-GA-Cys1-Tyr2-Gly3) inhibits the α-glucosidase enzyme by blocking its substrate binding site. AGEs-induced NO• inhibitors play an important role in controlling the inflammation associated with diabetes mellitus. The peptide conjugates 2-11 were also evaluated in vitro for AGEs-induced NO• inhibition using RAW 264.7 macrophage cell line. Our data revealed that conjugates 7-10 were the more potent AGEs-induced NO• inhibitors, comparable to standards rutin, and PDTC. The peptide conjugate 5 (a competitive inhibitor of α-glucosidase) also exhibited a strong inhibitory activity against AGEs-induced NO• production. Furthermore, peptide conjugates 2-11 were found non-cytotoxic to mouse fibroblast NIH-3T3, and murine macrophages RAW 264.7 cell lines. In conclusion, our data demonstrates that besides possessing strong α-glucosidase inhibition, the newly synthesized peptide conjugates also alleviated the AGEs-induced NO• production in RAW macrophages. Dual inhibition of α-glucosidase enzyme, and AGEs-induced NO• production by 18ß-GA-peptide conjugates qualify them for further research in anti-diabetic drug discovery.

In-vitro and in-vivo anticandidal activity of Trachyspermum ammi (L.) sprague seeds ethanolic extract and thymol-containing hexanes fraction.

Candida albicans is the causative agent of candidiasis in immunocompromised and diseased individuals. The current study was designed to evaluate the antifungal activity of Trachyspermum ammi (L.) Sprague seeds ethanolic extract and hexanes fraction against Candida albicans, both in-vitro and in-vivo. The minimal inhibitory concentration of hexanes fraction was found to be 225 µg/mL, as compared to the standard drug amphotericin B (200 µg/mL). For the treatment of cutaneous candidiasis in BALB/c mice model, the extract and fraction containing ointment was topically applied on flank of mice at various concentrations, and 90-100% recovery in mice was observed which was better than the standard drug, clotrimazole. The GC/MS analysis of hexanes extract indicated the presence of three major components, including (43.91%), O-cymene (25.53%), and γ-terpinene (22.64%). The results showed that T. ammi possess potential antifungal effect, and can be used for the development of novel antifungal agents after further pre-clinical and clinical trials.

A new metabolite from Cunninghamella blakesleeana-mediated biotransformation of an oral contraceptive drug, levonorgestrel.

Cunninghamella blakesleeana-mediated biotransformation of an oral contraceptive drug, levonorgestrel (1), yielded a new metabolite, 13ß-ethyl-17α-ethynyl-10,17ß-dihydroxy-4,6-dien-3-one (2), and two known metabolites 3 (13ß-ethyl-17α-ethynyl-10ß,17ß-dihydroxy-4-en-3-one), and 4 (13ß-ethyl-17α-ethynyl-6ß,17ß-dihydroxy-4-en-3-one) at an ambient temperature using aqueous media. Hydroxylation and dehydrogenation of compound 1 was observed during the bio-catalytic transformation. The structure of a new metabolite 2 was determined by 1H, 13C, and 2DNMR and HR-EIMS spectroscopic techniques.

Xuetonglactones A-F: Highly Oxidized Lanostane and Cycloartane Triterpenoids From Kadsura heteroclita Roxb. Craib.

Xuetonglactones A-F (1-6), six unreported highly oxidized lanostane- and cycloartane-type triterpenoids along with 22 known scaffolds (7-28) were isolated from the stems of Kadsura heteroclita (Roxb.) Craib. Compared with previous congeners, xuetonglactone A (1), possesses an unprecedented 20,21-α-epoxide, and xuetonglactone D (4) features an unusual 19-α-hydroperoxyl moiety. The structures and the absolute configurations of the compounds were established by extensive one- and two-dimensional NMR, and electronic circular dichroism (ECD) spectroscopic analysis, with those of 1 and 5 confirmed by single-crystal X-ray diffraction technique. Compounds 1 and 2 exhibited inhibition of iNOS activity in LPS-induced macrophages with IC50 values of 22.0, and 17.0 µg/mL, respectively. While compounds 6, 7, 8, and 24 showed potent cytotoxic activities against human cervical cancer cell lines (HeLa) with the IC50 values of 4.0, 5.8, 5.0, and 6.4 µM, and against human gastric cancer cells (BGC 823) with the IC50 values of 2.0, 5.0, 2.5, and 2.0 µM, respectively. Moreover, plausible biogenetic pathways of (1-6) were also proposed.

Protective effect of dietary polyphenol caffeic acid on ethylene glycol-induced kidney stones in rats.

Dietary polyphenol caffeic acid (1) has been reported for various pharmacological activities. The aim of the current study was to investigate the effect of caffeic acid (1) on ethylene glycol-induced renal stones in rats. For the study, male Wistar rats were divided into seven groups; normal, pathological, and standard drug controls, and preventive and curative groups. Normal control group received drinking water for 8 weeks. Pathological, standard drug, preventive, and curative groups received 0.75% ethylene glycol in drinking water for the induction of calcium oxalate stone formation, along with the regular diet. Standard drug group received Urocit-K by gavage from day 1, while preventive and curative groups received caffeic acid (1) by gavage at doses of 20 and 40 mg/kg on day 1 and day 14, respectively. At the end of the experiment, urine analysis and kidney histopathology were performed. Real-time PCR was performed to evaluate the renal expression of the most important genes involved in urolithiasis, i.e., osteopontin, Tamm-Horsfall, prothrombin fragment 1, and bikunin genes. The results indicated that in both the preventive and curative groups, treatment of rats with caffeic acid (1) significantly regulated the altered biochemical parameters, along with the remarkable reduction of calcium oxalate deposits in the kidneys, as compared to the pathological group. Treatment with compound 1 also resulted in down-regulation of the osteopontin gene, and up-regulation of the prothrombin fragment 1, Tamm-Horsfall, and bikunin genes. These results suggest that caffeic acid (1) can be further investigated for the prevention, and treatment of kidney stones.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Two new prenylated flavonoids from the roots of Berberis thunbergii DC.

Two new prenylated flavonoids, thunbergiols A (1) and B (2), along with three known compounds, chrysin (3), quercetin (4) and berberine (5) were obtained from the methanolic extract of roots of Berberis thunbergii DC. MS, NMR and other spectroscopic techniques were employed for their structural characterisation.

Microbial transformation of contraceptive drug etonogestrel into new metabolites with Cunninghamella blakesleeana and Cunninghamella echinulata.

Biotransformation of a steroidal contraceptive drug, etonogestrel (1), (13-ethyl-17ß-hydroxy-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3-one) was investigated with Cunninghamella blakesleeana and C. echinulata. Five metabolites 2-6 were obtained on incubation of 1 with Cunninghamella blakesleeana, and three metabolites, 2, 4, and 6 were isolated from the transformation of 1 with C. echinulata. Among them, metabolites 2-4 were identified as new compounds. Their structures were deduced as 6ß-hydroxy-11,22-epoxy-etonogestrel (2), 11,22-epoxy-etonogestrel (3), 10ß-hydroxy-etonogestrel (4), 6ß-hydroxy-etonogestrel (5), and 14α-hydroxy-etonogestrel (6). Compounds 1-6 were evaluated for various biological activities. Interestingly, compound 5 was found to be active against ß-glucuronidase enzyme with IC50 value of 13.97±0.12µM, in comparison to standard compound, d-saccharic acid 1,4-lactone (IC50=45.75±2.16µM). Intestinal bacteria produce ß-glucuronidase. Increased activity of ß-glucuronidase is responsible for the hydrolyses of glucuronic acid conjugates of estrogen and other toxic substances in the colon, which plays a key role in the etiology of colon cancer. Inhibition of ß-glucoronidase enzyme therefore has a therapeutic significance. Compounds 1-6 were also found to be non cytotoxic against 3T3 mouse fibroblast cell lines.

Microbial-catalysed derivatization of anti-cancer drug exemestane and cytotoxicity of resulting metabolites against human breast adenocarcinoma cell line (MCF-7) in vitro.

Structural transformation of anticancer drug exemestane (1) with fungi Cunninghamella blakesleeana (ATCC 8688A), Curvularia lunata (ATCC 12017), Aspergillus niger (ATCC 10549), and Gibberella fujikuroi (ATCC 10704) yielded eleven metabolites 2-12, in which 2 and 8 were identified as new. Their structures were characterized as 6-methylene-5α-androstane-3ß,16ß,17ß-triol (2), 17ß-hydroxy-6-methyleneandrosta-4-ene-3-one (3), 6α-spiroxirandrost-4-ene-3,17-dione (4), 6-methyleneandrosta-4-ene-3,17-dione (5), 6ß,17ß-dihydroxyandrost-4-en-3-one (6), 17ß-hydroxy-6α-spiroxirandrost-1,4-diene-3-one (7), 17ß-hydroxy-6α-hydroxymethylandrosta-1,4-dien-3-one (8), 6α-hydroxymethylandrosta-1,4-diene-3,17-dione (9), 17ß-hydroxy-6-methyleneandrosta-1,4-diene-3,16-dione (10), 6α-hydroxy-4-androstene-3,17-dione (11), and 6α-hydroxymethylandrost-4-ene-3,17-dione (12). Substrate 1, and its transformed products were evaluated for their cytotoxicity against breast cancer cell line (MCF-7). Compound 3 was found to be moderately active with an IC50 of 33.43±4.01µM, in comparison to the standard anti-cancer drug, doxorubicin (IC50=0.92±0.1µM).