key: cord-0003365-kzub5h7a authors: Alam, Perwez; Parvez, Mohammad K.; Arbab, Ahmed H.; Al-Dosari, Mohammed S. title: 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 date: 2017-03-10 journal: Pharm Biol DOI: 10.1080/13880209.2017.1300175 sha: 6dc7f0acaa45433001b3b0586094e88c0d400911 doc_id: 3365 cord_uid: kzub5h7a 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 60F(254)-RP18 and 60F(254), respectively. The methods were validated according to the ICH guidelines. Results: Well-separated and compact spots (R(f)) 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 (r(2)) 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. High-performance thin-layer chromatography (HPTLC) has recently become a conventional analytical tool for the quality-control of herbal drugs because of its low operation-cost, high sample-throughput and need for minimum sample clean-up . With HPTLC, qualitative and quantitative analyzes of multiple compounds can be done simultaneously by using small volume of mobile phase (Faiyazuddin et al. 2010) . The developed HPTLC chromatograms are useful in identification of biomarkers in various herbal formulations by comparing the fingerprints with standards . It is widely employed for the identification, purity testing, stability, dissolution or content uniformity of crude extracts of plant and animal origin, fermentation mix, drugs and excipients, including pharmaceutical, cosmetic and nutrient formulations (Alajmi et al. 2013) . Guiera senegalensis J.F. Gmel (Combretaceae) is one of the popular African folk medicine plants for treating a wide range of metabolic and infectious diseases (Bosisio et al. 1997; Somboro et al. 2011; Suleiman 2015) . The dried bitter leaves are the most important part of the plant, commonly sold in African markets as 'Cure all' medicine. An antitussive sirup (Nger), prepared from the G. senegalensis leaves, has been commercialized in Senegal (Sanogo et al. 1998) . Decoctions and various preparations of G. senegalensis are used to treat sexually transmitted, gastrointestinal, respiratory, fungal, bacterial and malarial diseases (Bosisio et al. 1997; Abubakar et al. 2000; Silva & Gomes 2003; Somboro et al. 2011; Akuodor et al. 2013; Suleiman 2015) . Moreover, the plant extract has shown to have antioxidative (Bouchet & Barrier 1998) , anti-inflammatory (Sombi e et al. 2011) and acaricidal (Osman et al. 2014) activities. Moreover, G. senegalensis was also reported to have antiviral activities against fowl pox (Lamien et al. 2005 ) and herpes (Silva et al. 1997) infections. Very recently, we demonstrated in vitro anti-hepatitis B virus (HBV) efficacy of G. senegalensis leaves extract (Parvez et al. 2016) . Further, among several groups of phytoconstituents, four flavonoids (catechin, myricitrin, rutin, and quercetin) (Bucar et al. 1996; Ficarra et al. 1997; Males et al. 1998) , two alkaloids (harman and tetrahydroharman or eleagnine) and one naphthyl butenone (guieranone A) are identified in the plant (Combier et al. 1997; Mahmoud & Sami 1997; Fiot et al. 2006 ). Recently, quercetin, a flavonoid is reported for its anti-HBV potential in vitro (Cheng et al. 2015) . Although biomarkers quercetin and rutin have been identified in G. senegalensis by HPLC (Males et al. 1998 ), a complete validated HPTLC method has not been reported yet for their quantitative analysis in G. senegalensis. Therefore, the present study intended to develop and validate normal phase (NP)-and reverse phase (RP)-HPTLC methods for quantifying the contents of rutin, quercetin, naringenin and gallic acid in the anti-HBV active extract of G. senegalensis leaves. Leaves of G. senegalensis locally known as 'Gubeish' were collected in March, 2015 from Kordofan state, Sudan. The plant material was authenticated by Prof. Ismail Mirghani, a taxonomist at the Forestry Research Center (FRC), Khartoum, Sudan, where a voucher specimen (No. 891) was deposited. Further authentication was confirmed at the herbarium of College of Pharmacy, King Saud University, Saudi Arabia. The leaves were shade dried at room temperature for 8 days. The dried leaves (50 g) were ground to fine powder using mortarpestle and extracted with 500 mL of 70% ethanol (Merck) for 24 h with intermittent shaking. The extraction process was repeated two times with fresh solvent. Then, extracts were pooled, filtered (Whatmann filter paper No. 1) and dried under reduced pressure using rotary evaporator (R-210, BUCHI). The biomarkers (rutin, quercetin, naringenin and gallic acid) were procured from Sigma Aldrich (USA). While AR grade chemicals viz., ethanol, acetonitrile, toluene, ethyl acetate and formic acid were procured from BDH (UK), HPLC grade ethanol and methanol were procured form Merk (Germany). For the analysis of samples and standards, glass-backed silica gel 60F 254 RP-18 plate (for RP-HPTLC) and glass-backed silica gel 60F 254 plate (for NP-HPTLC) were purchased from Merck (Germany). CAMAG Automatic TLC Sampler-4 (Switzerland) was used to apply the biomarkers and GSEE, band wise to the chromatographic plates and development was accomplished in automatic development chamber (ADC2) (Switzerland). The developed HPTLC Plates were then documented by CAMAG TLC Reprostar 3 and scanned by CAMAG CATS 4 (Switzerland). The HPTLC analysis of the biomarkers in GSEE was carried out on NP and RP-HPTLC plates (10 Â 10 cm) where the band size of each track was 6 mm wide and 8 mm apart. The samples were applied on the HPTLC plates (160 nL/s). The plates were developed in pre-saturated twin-trough glass chamber (20 Â 10 cm) at room temperature (25 ± 2 C) and humidity (60 ± 5%) using acetonitrile and water (4:6, v/v) for RP-HPTLC, and toluene, ethyl acetate and formic acid (6:4:0.8, v/v/v) for NP-HPTLC analysis. The developed and dried RP-HPTLC and NP-HPTLC plates were quantitatively analyzed at 360 and 275 nm in absorbance mode, respectively. Standard stocks of rutin, quercetin, naringenin and gallic acid were prepared in methanol (1 mg/mL). The stocks of rutin and quercetin were further diluted to furnish different concentrations ranging from 10 to 140 lg/mL. All the dilutions (10 lL, each) were applied through microliter syringe attached with the applicator on the RP-HPTLC plate to furnish the linearity range of 100-1400 ng/band for rutin and quercetin. Similarly, the dilutions of naringenin and gallic acid ranging from 10 to 120 lg/mL (10 lL, each) were applied to NP-HPTLC plate to furnish the linearity range of 100-1200 ng/band. Method of validation was carried out as per International Conference on Harmonization (ICH) guidelines for linearity range, limit of detection (LOD), limit of quantification (LOQ), precision, recovery as accuracy and robustness (ICH 2005) . The determination of LOD and LOQ was calculated using formula LOD ¼ 3.3(SD/S) and LOQ ¼ 10(SD/S), respectively, based on the standard deviation of the response (SD) and the slope (S) of the calibration curve. The precision (Intra-day and Inter-day) of the proposed HPTLC methods were evaluated for all biomarkers by performing replicate analysis (n ¼ 6) at three different concentration levels (low, medium and high) viz. 400, 600 and 800 ng/ band. The precision was recorded as Mean ± SD, %RSD and SEM of each calibration level. Recovery as accuracy studies involved the addition of a known amount of analyte to a sample, and determining the percentage of added analyte. For the biomarkers rutin, quercetin, naringenin and gallic acid, a known amount of 50, 100 and 150% of 200 ng, each was added and the recovery percentage of the spiked standards was estimated. The robustness of the proposed HPTLC methods were performed to analyze its capacity to remain unaffected by a small, but deliberate variations in mobile phase composition, mobile phase volume used for saturation and duration of saturation which indicates the reliability of the method during normal use. The robustness study was performed in replicate analysis (n ¼ 6) for all the markers at 300 ng/ band concentration. The results were evaluated in terms of SD, %RSD and SEM of peak area. In RP-HPTLC method, the mobile phases were prepared from acetonitrile: water (4:6, v/v) in different proportions (3.8:6.2, v/v and 4.2:5.8, v/v) and analyzed. In case of NP-HPTLC method, the different mobile phases (5.8:4.2:0.8 and 6.2:3.8:0.8, v/v/v) were prepared from toluene: ethyl acetate: gallic acid (6:4:0.8, v/v/v) and used for the analysis of markers to check its robustness. In addition to the minor variations in the mobile phases, the volume used for saturation was also varied from 20 to 18 and 22 mL. The duration of saturation also varied to 10 and 30 min from 20 min in the analysis. Results were expressed as mean ± SD. Total variation present in a set of data was estimated by one-way analysis of variance (ANOVA) followed by Dunnet's test. p < 0.01 was considered significant. The mobile phase used in RP-and NP-HPTLC analyses was selected by testing several compositions of different solvents. Of these, combination of acetonitrile and water (4:6, v/v) under chamber saturation condition was found to be the best mobile phase for the development and quantitative analysis of rutin and quercetin on RP-HPTLC plates. This method exhibited the clear separation of the two biomarkers along with the different constituents of GSEE ( Figure 1 ). On the other hand, for the analysis of naringenin and gallic acid on NP-HPTLC plates, the best mobile phase was the combination of toluene, ethyl acetate and gallic acid (6:4:0.8, v/v/v) which allowed their clear separation along with the different constituents of GSEE ( Figure 2 ). The optimized saturation time and volume of mobile phase for saturation were 20 min and 20 mL, respectively. The densitometric analysis of the biomarkers by the two HPTLC methods showed clearly separated compact, sharp, symmetrical and high resolution bands of rutin, quercetin, naringenin and gallic acid. While the bands of rutin and quercetin were obtained at R f 0.52 ± 0.006 and 0.23 ± 0.005, respectively (Figure 3) , those of naringenin and gallic acid were recorded at R f 0.56 ± 0.009 and 0.28 ± 0.006, respectively ( Figure 4) . The developed methods were thus, found quite selective with a good baseline resolution. Linearity of marker compounds rutin, quercetin, naringenin and gallic acid were validated by the linear regression equation and correlation coefficient. The seven-point calibration curve for rutin and quercetin was found linear in the range of 200-1400 ng whereas for naringenin and gallic acid it was in the range of 100-1200 ng. The observed regression equation (Y) and coefficient correlation (r 2 ) values for the biomarkers (Table 1 ) revealed a good linearity response for the developed methods. The LOD and LOQ for rutin, quercetin, naringenin, and gallic acid were also recorded ( Table 1) which indicated that the proposed method exhibits a good sensitivity for the simultaneous quantification of the above compounds. The %recovery, %RSD, and SEM were recorded in for rutin and quercetin (Table 2) , and naringenin and gallic acid (Table 3) for recoveries as accuracy study for the proposed methods. The intra-and inter-day precision (n ¼ 6) for the proposed RP-and NP-HPTLC methods were recorded as %RSD and SEM for rutin and quercetin (Table 4) , and for naringenin and gallic acid ( Table 5 ). The observed low values of %RSD and SEM indicated the good precision of both methods. Further, the low values of SD, %RSD and SEM obtained after introducing small deliberate changes in the two methods demonstrated the robustness of NP-HPTLC for rutin and quercetin (Table 6) , and RP-HPTLC for naringenin and gallic acid (Table 7) . The application of the proposed method was evaluated by applying this method for the quantitative analysis of rutin plus quercetin ( Figure 5 ) and naringenin plus gallic acid ( Figure 6 ) in GSEE. Notably, though the obtained peaks were near to each other in the two HPTLC methods, the corresponding bands were very clearly separated (Figures 3 and 4) . The calculated area of all peaks (AU) after their integration was peak-1: 1613.8; peak-2: 2310.4; peak-3: 105.3; peak-4 (quercetin): 5775.5; peak-5: 4389.5; peak-6: 29403.8; peak-7 (rutin): 17508.5 and peak-8: 1672.0 ( Figure 5) whereas it was peak-1: 614.6; peak-2: 112.1; peak-3: 578.5; peak-4: 128.6; peak-5 (gallic acid): 4564.8; peak-6: 10477.3); peak-7: 1707.8; peak-8: 1822.9; peak-9 (naringenin): 4819.5 and peak-10: 15116.4 ( Figure 6 ). The quantified contents of rutin, quercetin, naringenin and gallic acid in GSEE were 2.42, 1.53, 0.14 and 7.01 lg/mg of the dry weight of GSEE. This is the first report, demonstrating simple, accurate and rapid NP-and RP-HPTLC methods developed for the simultaneous quantification of antiviral biomarkers rutin, quercetin, naringenin and gallic acid in G. senegalensis. Guiera senegalensis, popularly known as 'Cure all' folk medicine in West and Central Africa, is used to treat various metabolic and infectious diseases (Aniagu et al. 2005; Diatta et al. 2007; Somboro et al. 2011; Suleiman 2015) . Though the therapeutic potential G. senegalensis is widely recognized, most of them are still at a preliminary level that need to be evaluated by scientific rationale and detailed research. In this report, we have developed NP-and RP-HPTLC methods for the quantification of four biomarkers: rutin, quercetin, naringenin and gallic acid in GSEE showing antiviral efficacy against hepatitis B (Parvez et al. 2016) . Rutin is a flavonoid that belongs to the family of vitamin C2, and is abundant in many vegetables, fruits and cereals. Rutin is a well-known antioxidant, anti-inflammatory and anticancer natural compound (Deschner et al. 1993; Guardia et al. 2001; Yanga et al. 2008; Lin et al. 2009) , and is sold commercially. Very recently, it has been demonstrated for promising antiviral efficacy against murine norovirus (MNV-1) in vitro (Ch eron et al. 2015) . In the present study, quantification of rutin (2.42 lg/mg) in the GSEE by RP-HPTLC method supports its possible role in the inhibition of HBV gene expressions and DNA replication. Quercetin is a flavonol found in natural products, especially in apples and onions (Hertog et al. 1993) . Quercetin is known to have multifaceted biological and therapeutic effects including antioxidative, anticancer, antimicrobial, anti-inflammatory, cardioprotective, and hepatoprotective activities (Harwood et al. 2007; Hern andez-Ortega et al. 2012; D'Andrea et al. 2015) . In addition, quercetin has in vitro antiviral activities against enveloped viruses such as meningovirus, Herpes simplex virus (HSV1), parainfluenza type 3, pseudorabies virus, respiratory syncytial virus, Sindbis virus (Mucsi 1984; Kaul et al. 1985; Vrijsen et al. 1988; Wleklik et al. 1988; Lamien et al. 2005; Choi et al. 2009; Chiow et al. 2016) , including HBV (Cheng et al. 2015) . Our quantitative analysis by validated RP-HPTLC method demonstrated quercetin (1.53 lg/mg) in the dry-weight of GSEE, therefore, strongly supports its anti-HBV activity. Naringenin is a flavanone found mainly in citrus fruits and tomatoes. Naringenin has many pharmacological properties including hypolipidemic, anti-hypertensive, anti-inflammatory, antioxidant, anti-fibrotic, and hepatoprotective activities (Yen et al. 2009; Cho et al. 2011; Hermenean et al. 2014; Motawi et al. 2014; Chtourou et al. 2015) . Interestingly, naringenien has been also reported for its antiviral potential against HCV through blocking the assembly of intracellular viral particles (Goldwasser et al. 2011 ). Our quantification of naringenin (0.14 lg/mg), though at a low level in SSEE by NP-HPTLC method indicates its possible role in inhibiting HBV life cycle. Gallic acid is a phenolic compound obtained from plants, fruits and vegetables. Gallic acid and structurally related compounds possess many potential therapeutic properties including anti-cancer, anti-inflammatory and anti-microbial effects (Inoue et al. 1995; You & Park 2001; Ow & Stupans 2003; Kim 2007; Chen et al. 2009; Ji et al. 2009; Deng et al. 2014; Oyedeji et al. 2014; Xiaoyong & Luming 2014) . In addition, gallic acid exhibited antiviral activities against enterovirus-71 (Choi et al. 2010 ), Herpes simplex virus type 1 (HSV-1), anti-human immunodeficiency virus (Kratz et al. 2008 ) and hepatitis C virus (HCV) (Govea-Salas et al. 2016) . In our quantitative analysis by validated NP-HPTLC method, gallic acid (7.01 lg/mg) was estimated the most abundant biomarker in GSEE. Identification of gallic acid known for antiviral potential is in line with its recently reported anti-HBV activity. As discussed above, the four biomarkers, rutin, quercetin, nargennin and gallic acid have antiviral potentials against a variety of biologically related but genetically RNA and DNA viruses. Of these, HSV, HIV and HBV are enveloped (coated) viruses but unlike HSV and HIV, HBV is a DNA virus. Notably, HBV uniquely replicates its DNA genome via an RNA intermediate through reverse-transcription similar to RNA viruses. Interestingly therefore, almost all potential nucleos(t)ide-based antiviral agents developed for HSV and HIV, have been effective against HBV. Moreover, HCV, an enveloped RNA virus, does not share the antiviral regimens of HBV (except, the cytokine interferon). Therefore, the effectiveness of rutin, quercetin, nargennin and gallic acid against these viruses could be explained by considering the common inhibitory mechanism either targeting viral envelopes or reverse-transcriptases. Nevertheless, addressing this issue is out of the scope of the present study. Notably, except rutin, quercetin, naringenin and gallic acid, we could not study the other antiviral biomrkers in GSEE due to some limitations. There is a very high possibility of presence of other potential biomarkers in G. senegalensis that needs further analysis. Our quantitative analysis of four antiviral biomarkers by the RPand NP-HPTLC methods furnished gallic acid (7.01 lg/mg) the most abundant antiviral biomarker compared to rutin (2.42 lg/ mg), quercetin (1.53 lg/mg) and naringenin (0.14 lg/mg) in G. senegalensis leaves. To the best of our knowledge, this is the first report demonstrating validation of simple, accurate and sensitive NP-and RP-HPTLC methods for the separation of different phytoconstituents and simultaneous quantification of antiviral biomarkers in G. senegalensis. In addition, our data scientifically endorses the traditional knowledge of G. senegalensis in folk medicine, including its anti-HBV activities. The developed methods could be therefore, employed in the standardization and quality-control of herbal preparations containing therapeutic biomarkers. In vitro snake venom detoxifying action of the leaf extract of Guiera senegalensis Gastroprotective effect of the aqueous leaf extract of Guiera senegalensis in Albino rats Anti-diarrhoeal and ulcer-protective effects of the aqueous root extract of Guiera senegalensis in rodents Stability-indicating densitometric high-performance thinlayer chromatographic method for the quantitative analysis of biomarker naringin in the leaves and stems of Rumex vesicarius L Quantification of bioactive marker b-amyrin by validated high-performance thin-layer chromatographic densitometric method in different species of Maytenus grown in Kingdom of Saudi Arabia Guiera senegalensis JF Gmelin (Combretaceae): Biological activities and chemical investigation Radical scavenging activity and antioxidant properties of tannins from Guiera senegalensis (Combretaceae) Flavonoids of Guiera senegalensis Gallic acid, a major component of Toona sinensis leaf extracts, contains a ROS-mediated anti-cancer activity in human prostate cancer cells Inhibition of hepatitis B virus replication by quercetin in human hepatoma cell lines Repurposing of rutin for the inhibition of norovirus replication Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection Naringenin reduces cholesterol-induced hepatic inflammation in rats by modulating matrix metalloproteinases-2, 9 via inhibition of nuclear factor jB pathway Dietary naringenin increases hepatic peroxisome proliferators-activated receptor a protein expression and decreases plasma triglyceride and adiposity in rats Anti-Human rhinovirus activity of gallic acid possessing antioxidant capacity Antiviral activity of quercetin 7-rhamnoside against porcine epidemic diarrhea virus Alcaloïdes du Guiera senegalensis Lam. Plantes M ed Phytoth er Quercetin: a flavonol with multifaceted therapeutic applications Influences of ripening stages and extracting solvents on the polyphenolic compounds, antimicrobial and antioxidant activities of blueberry leaf extracts The effect of dietary quercetin and rutin on AOM-induced acute colonic epithelial abnormalities in mice fed a high-fat diet Experimental evidence of cough activity of total alkaloids from Guiera senegalensis Lam. in guinea pig Chromatographic analysis of trans and cis-citral in lemongrass oil and in a topical phytonanocosmeceutical formulation and validation of the method Isolation and characterization of Guiera senegalensis J.F.Gmel. active principles Phytochemical and pharmacological study of roots and leaves of Guiera senegalensis Naringenin inhibits the assembly and long-term production of infectious hepatitis C virus particles through a PPAR-mediated mechanism Gallic acid decreases hepatitis C virus expression through its antioxidant capacity Anti-inflammatory properties of plant flavonoids. Effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/ carcinogenic properties Antioxidant and hepatoprotective effects of naringenin and its b-cyclodextrin formulation in mice intoxicated with carbon tetrachloride: a comparative study Intake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands Quercetin improves hepatic fibrosis reducing hepatic stellate cells and regulating pro-fibrogenic/anti-fibrogenic molecules balance Selective induction of cell death in cancer cells by gallic acid Gallic acid induces apoptosis via caspase-3 and mitochondrion-dependent pathways in vitro and suppresses lung xenograft tumor growth in vivo Antiviral effect of flavonoids on human viruses Antimelanogenic and antioxidant properties of gallic acid Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate Inhibition of fowlpox virus by an aqueous acetone extract from galls of Guiera senegalensis J. F. Gmel (Combretaceae) Rutin inhibits the proliferation of murine leukemia WEHI-3 cells in vivo and promotes immune response in vivo 5-Methyldihydroflavasperone, a dihydronaphthopyran from Guiera senegalensis Flavonoids of Guiera senegalensis J.F. Gmel.-thin-layer chromatography and numerical methods Effect of simvastatin and naringenin coadministration on rat liver DNA fragmentation and cytochrome P450 activity: an in vivo and in vitro study Combined antiviral effects of flavonoids and 5-ethyl-2'-deoxyuridine on the multiplication of herpesviruses Acaricidal properties of two extracts from Guiera senegalensis J.F. Gmel. (Combretaceae) against Hyalomma anatolicum (Acari: Ixodidae) In vitro antimicrobial and antioxidant analysis of gallic acid from the leaves of Ludwigia abyssinica A. Rich Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes In vitro evaluations of anti-hepatitis B activities of 60 medicinal plants extracts The antitussive activity of Guiera senegalensis J.F.Gmel. (Combretaceae) Quantification of physiologically available glycyrrhizin in anti-stress herbal formulations by validated HPTLC method Plant extracts antiviral activity against Herpes simplex virus type 1 and African swine fever virus Guieranone A, a naphthyl butenone from the leaves of Guiera senegalensis with antifungal activity An ethnobotanical and phytochemical study of the African medicinal plant Guiera senegalensis J.F. Gmel Antioxidant and anti-inflammatory activities from galls of Guiera senegalensis An ethnobotanical survey of medicinal plants used by communities of Northern Kordofan region Antiviral activity of flavones and potentiation by ascorbate Structural basis for antiviral activity of flavonoids-naturally occurring compounds In vitro antioxidant properties of rutin Naringenin-loaded nanoparticles improve the physicochemical properties and the hepatoprotective effects of naringenin in orally-administered rats with CCl 4 -induced acute liver failure The effects of mitogen-activated protein kinase inhibitors or small interfering RNAs on gallic acid-induced HeLa cell death in relation to reactive oxygen species and glutathione Phenolic constituents, antimicrobial and antioxidant properties of blueberry leaves (V5) The authors declare that they do not have conflict of interest. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RG-1435-053.