Isolation and Characterization of Two Chalcone Derivatives with Anti-Hepatitis B Virus Activity from the Endemic Socotraen Dracaena cinnabari (Dragon's Blood Tree).

Hepatitis B virus (HBV) infection is prevalent and continues to be a global health concern. In this study, we determined the anti-hepatitis B virus (HBV) potential of the Socotra-endemic medicinal plant Dracaena cinnabari and isolated and characterized the responsible constituents. A bioassay-guided fractionation using different chromatographic techniques of the methanolic extract of D. cinnabari led to the isolation of two chalcone derivatives. Using a variety of spectroscopic techniques, including 1H-, 13C-, and 2D-NMR, these derivatives were identified as 2,4'-dihydroxy-4-methoxydihydrochalcone (compound 1) and 2,4'-dihydroxy-4-methoxyhydrochalcone (compound 2). Both compounds were isolated for the first time from the red resin (dragon's blood) of D. cinnabari. The compounds were first evaluated for cytotoxicity on HepG2.2.15 cells and 50% cytotoxicity concentration (CC50) values were determined. They were then evaluated for anti-HBV activity against HepG2.2.15 cells by assessing the suppression of HBsAg and HBeAg production in the culture supernatants and their half maximum inhibitory concentration (IC50) and therapeutic index (TI) values were determined. Compounds 1 and 2 indicated inhibition of HBsAg production in a dose- and time-dependent manner with IC50 values of 20.56 and 6.36 µg/mL, respectively.

Bioassay-guided isolation of anti-hepatitis B virus flavonoid myricetin-3-O-rhamnoside along with quercetin from Guiera senegalensis leaves.

Recently, we have shown in vitro anti-hepatitis B virus (HBV) activity of G. senegalensis J.F. Gmel leaves, and Identified quercetin and other flavonoids by HPTLC. Here we report bioassay-directed fractionation of G. senegalensis leaves using column chromatography and isolation of two flavonoinds from the n-butanol fraction, their structure determination (1H NMR, 13C NMR and 2D-NMR) and assessment of antiviral activities (HBsAg and HBeAg assay) in HBV-reporter HepG2.2.2.15 cells. Further molecular docking was performed against HBV polymerase (Pol/RT) and capsid (Core) proteins as well as host-receptor sodium taurocholate co-transporting polypeptide (NTCP). The two isolated bioactive compounds were identified as quercetin and myricetin-3-O-rhamnoside. Quercetin significantly inhibited synthesis of HBsAg and HBeAg by about 60% and 62%, respectively as compared to myricetin-3-O-rhamnoside by 44% and 35%, respectively. Molecular docking of the two anti-HBV flavonoids revealed their higher binding affinities towards Pol/RT than Core and NTCP. In conclusion, this is the first report on anti-HBV active myricetin-3-O-rhamnoside along with quercetin isolated from G. senegalensis leaves. Their possible mode of anti-HBV activities are suggested through binding with viral Pol/RT and Core as well as host NTCP proteins.

Hepatoprotective effect of Solanum surattense leaf extract against chemical- induced oxidative and apoptotic injury in rats.

BACKGROUND: Of over 35 Saudi plants traditionally used to treat liver disorders, majority still lack scientific validations. We therefore, evaluated the anti-oxidative, anti-apoptotic and hepatoprotective potential of Solanum surattense leaves total ethanol-extract (SSEE). METHODS: The cytoprotective (4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide/ MTT assay) and anti-apoptotic (caspase-3/7) potential of SSEE (25-200 µg/mL) were assessed in cultured HepG2 cells against dichlorofluorescein (DCFH)-induced toxicity. The hepatoprotective salutation of SSEE (100 and 200 mg/kg.bw/day) in carbon tetrachloride (CCl4)-intoxicated rats was evaluated by serum biochemistry and histopathology. The anti-oxidative activity of SSEE (31.25-500 µg/mL) was tested by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic acid bleaching assays. Also, SSEE was subjected to qualitative phytochemical analysis, and standardized by validated high-performance liquid chromatography (HPTLC). RESULTS: SSEE at doses 50, 100 and 200 µg/mL showed HepG2 cell proliferative and protective potential by about 61.0, 67.2 and 95%, respectively through inhibition of caspase-3/7 against DCFH-toxicity. In CCl4-injured rats, SSEE (200 mg/kg) significantly (P < 0.001) normalized serum transaminases, alkaline phosphatase, bilirubin, cholesterol, triglycerides, and total protein, including tissue malondialdehyde and nonprotein sulfhydryls levels, supported by the liver histopathology. SSEE further showed strong in vitro anti-oxidative and anti-lipid peroxidative activities, evidenced by the presence of alkaloids, flavonoids, tannins, sterols and saponins. Identification of ß-sitosterol (3.46 µg/mg) strongly supported the anti-oxidative and hepatoprotective salutation of SSEE. CONCLUSION: Our findings suggest the therapeutic potential of S. surattense against chemical-induced oxidative stress and liver damage. However, isolation of the active principles and elucidation of mechanism of action remain to be addressed.

The in vitro and in vivo anti-hepatotoxic, anti-hepatitis B virus and hepatic CYP450 modulating potential of Cyperus rotundus.

In the present study we investigated the hepatotoprotective, hepatitis B virus (HBV) inhibitory and hepatic CYP450 enzyme (CYP3A4) modulatory potential of Cyperus rotundus rhizome fractions. The crude ethanol-extract, including different organic and aqueous fractions were tested for in vitro cytoprotection on HepG2 cells (MTT assay), followed by in vivo evaluation in Wistar rats (serum biochemistry and lipid profile). The in vitro anti-HBV activity was tested on HepG2.2.15 cells (HBsAg and HBeAg Elisa). Of these, the n-butanol and aqueous fractions showed the most promising, dose-dependent hepatoprotection in DCFH-injured HepG2 cells. Further, in CCl4-injured rats, oral administration of C. rotundus (100 and 200 mg/kg·bw/day) significantly normalized serum markers of healthy liver function (SGOT, SGPT, GGT, ALP and bilirubin) and lipid profile (cholesterol, HDL, LDL, VLDL, TG and MDA), including tissue NP-SH and TP levels. Compared to other fractions, the ethyl acetate, n-butanol and aqueous fractions exhibited the best inhibitory effects on viral HBsAg and HBeAg secretions in dose- and time-dependent manner. In addition, reporter gene assay (Dual-luciferase) of transfected HepG2 cells showed mild activation of nuclear PXR-mediated CYP3A4 gene by the three active fractions. Taken together, C. rotundus showed very promising hepatoprotective and anti-HBV potential in experimental settings. In addition, this is the first report on modulation of CYP3A4 by C. rotundus that suggests its safe consumption in relation to drug metabolism and efficacy. Our data could therefore, provide the basis for the ethnobotanical medicinal use of C. rotundus in metabolic liver disorder and hepatitis B patients.

Analysis of antioxidative and antiviral biomarkers ß-amyrin, ß-sitosterol, lupeol, ursolic acid in Guiera senegalensis leaves extract by validated HPTLC methods.

Guiera senegalensis J.F. Gmel is a broad-spectrum African folk- medicinal plant, having activities against fowlpox and herpes viruses. Very recently, we have shown the anti-hepatitis B vius (HBV) potential of G. senegalensis leaves extract (GSLE). Here, we report the antioxidative and hepatoprotective efficacy of GSLE, including HPTLC quantification of four biomarkers of known antioxidative and antiviral activities. In cultured liver cells (HuH7) GSLE attenuated DCFH-induced oxidative stress and cytotoxicity. This was supported by in vitro DPPH radical-scavenging and ß-carotene-linoleic acid bleaching assays that showed strong antioxidant activity of GSLE. Further, two simple and sensitive HPTLC methods (I and II) were developed and validated to quantify ß-amyrin, ß- sitosterol, lupeol, ursolic acid in GSLE. While HPTLC-I (hexane: ethylacetate; 75:25; v/v) enabled quantification of ß-amyrin (Rf = 0.39; 20.64 µg/mg) and ß-sitosterol (Rf = 0.25; 18.56 µg/mg), HPTLC-II (chloroform: methanol; 97:3; v/v) allowed estimation of lupeol (Rf = 0.47; 6.72 µg/mg) and ursolic acid (Rf = 0.23; 5.81 µg/mg) in GSLE. Taken together, the identified biomarkers strongly supported the antioxidant and anti-HBV potential of GSLE, suggesting its activity via abating the oxidative stress. To our knowledge, this is the first report on HPTLC analysis of these biomarkers in G. senegalensis that could be adopted for standardization and quality-control of herbal-formulations.

Quantitative analysis of rutin, quercetin, naringenin, and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis.

CONTEXT: Guiera senegalensis J.F. Gmel (Combretaceae) is a folk medicinal plant used in various metabolic and infectious diseases. In addition to its antiviral activities against herpes and fowlpox, the anti-HBV efficacy is very recently reported. OBJECTIVE: To develop and validate simple, sensitive RP-/NP-HPTLC methods for quantitative determination of biomarkers rutin, quercetin, naringenin, and gallic acid in the anti-HBV active G. senegalensis leaves ethanol-extract. MATERIALS AND METHODS: RP-HPTLC (rutin & quercetin; phase- acetonitrile:water, 4:6) and NP-HPTLC (naringenin & gallic acid; phase- toluene:ethyl acetate:formic acid, 6:4:0.8) were performed on glass-backed silica gel plates 60F254-RP18 and 60F254, respectively. The methods were validated according to the ICH guidelines. RESULTS: Well-separated and compact spots (Rf) of rutin (0.52 ± 0.006), quercetin (0.23 ± 0.005), naringenin (0.56 ± 0.009) and gallic acid (0.28 ± 0.006) were detected. The regression equations (Y) were 12.434x + 443.49, 10.08x + 216.85, 11.253x + 973.52 and 11.082x + 446.41 whereas the coefficient correlations (r2) were 0.997 ± 0.0004, 0.9982 ± 0.0001, 0.9974 ± 0.0004 and 0.9981 ± 0.0001, respectively. The linearity ranges (ng/spot) were 200-1400 (RP-HPTLC) and 100-1200 (NP-HPTLC). The LOD/LOQ (ng/band) were 33.03/100.1 (rutin), 9.67/29.31 (quercetin), 35.574/107.8 (naringenin), and 12.32/37.35 (gallic acid). Gallic acid (7.01 µg/mg) was the most abundant biomarker compared to rutin (2.42 µg/mg), quercetin (1.53 µg/mg) and naringenin (0.14 µg/mg) in the extract. CONCLUSION: The validated NP-/RP-HPTLC methods were simple, accurate, and sensitive for separating and quantifying antiviral biomarkers in G. senegalensis, and endorsed its anti-HBV activity. The developed methods could be further employed in the standardization and quality-control of herbal formulations.

Comparative study of antioxidant activity and validated RP-HPTLC analysis of rutin in the leaves of different Acacia species grown in Saudi Arabia.

The present study assessed the comparative antioxidant potential of the ethanol extract (EE) of leaves of four Acacia species (Acacia salicina, AS; Acacia laeta, AL; Acacia hamulosa AH; and Acacia tortilis, AT) grown in Saudi Arabia, including RP-HPTLC quantification of antioxidant biomarker rutin. In vitro DPPH radical scavenging and ß-carotene-linoleic acid bleaching assays showed the promising antioxidant activities of Acacia extracts: ASEE (IC50: 60.39 and 324.65 µg/ml) >ALEE (IC50: 217.06 and 423.36 µg/ml) >ATEE (IC50: 250.13 and 747.50 µg/ml) >AHEE (IC50: 255.83 and 417.28 µg/ml). This was comparable to rutin tested at 500 µg/ml. Further, a RP- HPTLC densitometric method was developed (acetonitrile:water; 6:4; v/v) using glass-backed RP-18 silica gel F254 plate, and scanned at UV max 254 nm. The method was validated as per the ICH guidelines. Analysis of the validated RP-HPTLC displayed an intense peak (Rf = 0.65 ± 0.004) of rutin that was estimated (µg/mg dry weight) to be highest in ASEE (10.42), followed by ALEE (2.67), AHEE (1.36) and ATEE (0.31). Taken together, presence of rutin strongly supported the high antioxidant property of the tested Acacia species, especially Acacia salicina. The developed RP-HPTLC method therefore, affirms its application in the quality control of commercialized herbal drugs or formulation containing rutin.

In-Vitro Anticancer Evaluation of Some Novel Thioureido-Benzensulfonamide Derivatives.

A novel series of sulfonamide derivatives (14 compounds) bearing thiourea moieties were efficiently synthesized and evaluated for their possible in vitro anticancer activity against four human tumor cell lines. The results indicated that compound 6 was the most potent, showing effectiveness on all the tested cell lines. Compounds 7 and 10 also showed promising results.

Attitude and Practice of Pharmacy Students Toward Traditional Herbal Medicines, Sudan.

Purpose: Traditional herbal medicines (THMs) are widely used in Sudan, but there is scarcity of research on pharmacy students' attitudes and utilization of THMs. This study aims to assess the attitudes and practices of pharmacy students toward THM. Methods: A descriptive cross-sectional study was conducted among randomly selected undergraduate pharmacy students at the University of Khartoum (Sudan). Data were collected using a validated self-administered questionnaire adapted from previous studies. A statistical package for the social sciences was used for data analysis. Results: Among the 264 respondents, the average age was 21.8 years, and 84.8% of them were females. Nearly 85% of respondents were residents of Khartoum. The overall respondent's attitudes towards THMs were positive, it ranged from high (19.7%), moderate (79.9%). About 87.8% of respondents are interested in learning more about THMs. In addition, 36.4%, and 50.4% of respondents strongly agreed or agreed that THMs should be officially licensed and controlled. A 17.8% and 63.3% of respondents, respectively, reported using traditional herbal remedies either always or occasionally. Supermarkets (35.2%) and friends/family (36.4%) were the main sources of THMs for the participants, and oral administration was the most used route (87.1%). More than 90% of respondents said they would suggest THMs to others, and the most common justifications for doing so were their accessibility (46.6%) and affordability (16.3%). The most widely used herbs were Mentha spicata, Zingiber officinale, Acacia nilotica, Hibiscus sabdariffa, and Syzygium aromaticum. Data revealed a significant association between attitude levels and respondents' gender (p=0.046), year of study (p=0.000), and residence (p=0.017). Conclusion: Most undergraduate pharmacy students had a positive attitude and utilized THMs. Development of effective educational initiatives, research, and regulations are recommended to ensure the appropriate utilization of traditional herbal remedies.

Knowledge and Attitude of Community Pharmacists Toward Cow Milk Protein Allergy Complications and Treatment in Children: A Cross-Sectional Study in Sudan.

Background: Cow's milk protein allergy is the most frequent type of food allergy in newborns and young children. It affects numerous body systems. A community pharmacist possessing adequate knowledge and appropriate practices is crucial for the management of the disease. Objectives: : This study aimed to assess the knowledge and attitude of community pharmacists toward the complications and treatment of cow milk protein allergy. Methods: A descriptive cross-sectional study was conducted among 332 randomly selected community pharmacists in the Capital of Sudan (Khartoum state). Data were collected using a validated and internally consistent (Cronbach Alpha = 0.712) self-administered questionnaire Descriptive statistics and Chi-square analysis were conducted using Statistical Package for the Social Sciences version 25. Results: The response rate was 95.8%, and about 47.8% of the population had 1-5 years of experience as community pharmacists, and 37.4% of them had a post-graduate degree. Only 30.8% of respondents had good knowledge, while 40.6% had poor knowledge, and 28.6% had scored as having fair knowledge of complications of Cow's milk protein allergy. Regarding knowledge toward treatment, 65.1% of respondents had a poor knowledge, 24.5% had fair knowledge, and only 10.4% had good knowledge. Overall, 39.62% of participants had a positive attitude, and 60.38% had a negative attitude toward this disease. Data analysis showed no significant association between the sociodemographic characteristics of respondents and their knowledge and attitude. Conclusion: Community pharmacists in Khartoum State had inadequate knowledge and attitude about the complications and treatment of cow milk protein allergy. As a result, we recommend the implementation of strategies to raise pharmacists' knowledge and attitude.