Department of Pharmacognosy

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    Pharmacological Screening of Selected Sudanese Medicinal Plants for Analgesic, Antinociceptive, Antipyretic and Sedative Effects
    (University of Khartoum, 2022) Muna Gafaar Idris Nabag
    Abstract ● Background: Plant-based therapies are widely used in Sudan and around the world. Acacia gerrardii Benth (السلجم), Asparagus flagellaris (Kunth) Baker (السريح) and Tephrosia uniflora Pers (عرق التيس) from Eastern Nuba mountains are frequently used in folk medicine for many ailments including pain. The current study aimed to assess the analgesic, antinociceptive, antipyretic and sedative effects of the above-mentioned plants. Acute oral toxicity of the extracts was also investigated by determining the LD50. ● Methods: Dichloromethane and ethanolic extracts of the leaves and roots of the selected plants were tested in albino rats of both sexes weighing (110- 210g). For each test 17 groups of rats ( n=6) were used, the doses were (100, 200, and 400 mg/kg orally) from each extract in each of the tests and normal saline 10ml/kg used as control. Extracts were investigated for analgesic and antinociceptive activities using the acetic acid-induced writhing test, formalin-induced paw licking test, tail flick method and hot plate method. Morphine (5mg/kg) was used as standard drug. The mechanism of action elucidated through pre-treatment with naloxone (1 mg/kg) in hot plate test for the ethanolic extracts of A. flagellaris. Sedative effect was examined by open field test and hole board tests and diazepam (1mg/kg) was used as standard drug. The antipyretic effect of the selected plants was evaluated by Brewer’s yeast test, and paracetamol (150mg/kg) was the standard drug. Ethanolic extracts were tested for acute oral toxicity. Firstly three groups (n=3) were fed with single oral dose of the extract (10, 100, and 1000 mg/kg), when no mortality after observation for 24 hours a second test carried for three groups (n=1) for doses of (1,600, 2,900, and 5000 mg/kg). The animals were closely observed for any general and behavioral changes for 24 hours, and then LD50 value was calculated. Data analysis: Results were calculated as mean ± standard error of the mean, by using one-way ANOVA flowed by Tukey post hoc test in SPSS. P value (p<0.05) was considered significant. ● Results: Ethanolic extract A. flagellaris leaves (400mg/kg) recorded 87.92% (p≤ 0.001) in acetic acid writhing, 86.51% and 79.89% (p≤ 0.001) in early and late phases of formalin test respectively, while ethanolic extract A. flagellaris roots exhibited the highest retention 14.97±0.19 seconds at 120 minutes (p≤ 0.001) in the tail flick test. In hot plate test, the highest significant (p≤ 0.001) latency was 27.31±0.79 seconds displayed by the ethanolic extract A. flagellaris leaves with latency being reduced to time close to that of the control normal saline when rats pretreated with naloxone. When the sedative effect examined by the open-field method; ethanolic extracts of T. uniflora roots (400mg/kg) had significantly (p≤ 0.001) decreased the squares crossed to 2.80±0.20 square at 120 minutes. For hole board test; ethanolic extracts of A. gerrardii (leaves and roots) and T. uniflora roots produced a significant (p≤ 0.001) decrease of head dips of 0.83±0.30 dips at 120 minutes for the dose (400mg/kg). Similarly, the dichloromethane extract Acacia gerrardii leaves (400mg/kg) produces the lowest significant (p≤ 0.001) decrease of head dips (0.830.±0.65 head dip). In Brewer’s yeast test, the dichloromethane leaves extract of A. gerrardii (200mg/kg) significantly (p≤ 0.001) decreased the rats’ rectal temperature, to 35.6Cº± 0.12 after 5 hours. Similar response was produced with dichloromethane extract of T. uniflora roots (400mg/kg) with significant (p≤0.001) reduction in rectal temperature to 35.57Cº±0.21. Gas Chromatography/Mass Spectrometric (GC/MS) analysis of the ethanolic extracts of A. flagellaris leaves and roots displayed many compounds with reported analgesic and antinociceptive activity. Acute oral toxicity test for the ethanolic extracts of A. gerrardii and A. flagellaris revealed no mortality at any dose, while T. uniflora roots had mortality at 5000mg/kg, with LD50 calculated to be 3800 mg/kg. ● Conclusion: Ethanolic extracts of leaves and roots of A. flagellaris showed promising analgesic and antinociceptive activity, while those of A. gerrardii and T. uniflora exhibited significant sedative and antipyretic activities. The tested ethanolic extracts exhibited high oral safety profile. Recommendations: Further studies are needed to fully characterize the responsible active compounds present in A. flagellaris extracts in order to proceed for preparing pharmaceutical form. A. gerrardii and T. uniflora need further studied to identify the active compounds and the mechanism of action.
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    Phytochemical, Antioxidant and Anti-acetylcholinesterase Screening of Some Selected Plants from Sudan
    (University of Khartoum, 2021) Mohamed Abakar Maaz Mohamed
    Background: Antioxidants are substances that significantly decrease the harmful effects of free radical oxygen or nitrogen reactive species. There are active ongoing researches to identify new antioxidants and anti-cholinesterase products from natural sources for the prevention of Alzheimer’s disease. Methods: In vitro DPPH, superoxide and iron chelating antioxidant along with anti-cholinesterase activities of ethanol, petroleum ether, chloroform, ethyl-acetate and n-butanol extracts obtained from three plants from Sudan were evaluated, namely Ambrosia maritema (Sea Ragweed), Eclipta prostrata (False daisy), and Geigeria alata (Wing vomidaisy), (Family: Asteraceae). The phytoconstituents’ anti-cholinesterase activity was predicted manipulating molecular docking tools. Results: The highest DPPH radical scavenging activity was demonstrated by both ethyl acetate and n-butanol fractions obtained from G. alata with percentages of inhibition 91 ±0.02% and 90 ±0.02% (IC50 = 22±0.01 and 66 ±0.02 µg/ml) respectively. Ethyl acetate fraction obtained from A. maritima has shown a potent superoxide radical scavenging activity with a percentage of inhibition 94±0.01% and IC50 = 48 ±0.01 µg/ml. Variety of secondary plant metabolites have been detected in the obtained fractions. Pointing at the in-vitro AChE inhibitory activity results, the ethyl acetate fraction obtained from G. alata has shown the highest AChE inhibitory activity (78% inhibition, IC50 0.246 ± 0.02 mg/ml) compared to the other plants’ extracts and the standard control inhibitory activities (eserine: 70%inhibition, IC50 0.00032 ± 0.01 mg/ml). Data result also indicated that the fraction has a significant (P < 0.05) value than the control group. Among the nine GC/MS detected phytoconstituents and twenty reported phytoconstituents docked against 4EY7 and 6WVC rAChEs X-ray crystallographic structures, eighteen compounds exhibited Vs-scores higher than both, the co-crystalized ligand and the standard positive control Vs-scores’. Quercetin was ranked top on the list of poses (LF Rank Score). Yet in terms of molecular docking, predicted pharmacokinetic and toxicity profiles collectively, hispidulin was the best (Vs-score: -9.091 and -9.226, respectively). Conclusion: Ethyl-acetate fraction obtained from G. alata demonstrated promising in-vitro antioxidant and AChE inhibitory activities. The flavonoid hispidulin reported in the same fraction presumed to be the lead compound in the fraction with a remarkable predicted enzyme inhibition, pharmacokinetic and toxicity profiles. Noteworthy, no previous studies were found in the literature concerning AChE inhibition activity of the three plants. Many desperate phytoconstituents, mostly, phenolic derivatives were detected in the screened plants’ extracts. This work provides a promising insight into the importance of further studies on the studied plants’ extracts towards isolation and characterization of AChE inhibitory phytoconstituents. المقدمـــــة : مضادات الأكسدة هي المواد التي تقلل بشكل كبير من الآثار الضارة للجذور الأكسجينية أو النيتروجينة الحرة. هناك العديد من الأبحاث الجارية لإيجاد مضادات أكسدة و مثبطات إزيم الكولينستيراز جديدة من المصادر الطبيعية للوقاية من مرض الزهايمر. الطُــــــرق: تمت دراسة النشاط المضاد للأكسدة و نشاط تثبيط إنزيم أسيتيل كولينستراز لمستخلصات الإيثانول ، أثير البترول, الكلوروفورم, مستخلصات إيثيل أسيتات و البيوتانول التي تم الحصول عليها من ثلاثة نباتات مختارة نامية في السودان و تنتمي للعائلة النجمية و هي الدمسيسة ، تمر الغنم ، و القدقاد. كما درس النشاط المضاد للكولينستريز للمكونات النباتية باستخدام برامج الكيمياء الحاسوبية. النتائــــج: وجد أن أعلى نشاط لـتثبيط جذور DPPH بواسطة كل من كسور أسيتات الإيثيل و n- بيوتانول التي تم الحصول عليها من G. alata بنسب مئوية للتثبيط 91 ± 0.02٪ و 90 ± 0.02٪ (IC50 = 22 ± 0.01 و 66 ± 0.02 ميكروغرام / مل) على التوالي. أظهر جزء أسيتات الإيثيل الذي تم الحصول عليه من A. maritima نشاطًا قويًا في الكسح الجذري للأكسيد الفائق مع نسبة تثبيط 94 ± 0.01٪ و IC50 = 48 ± 0.01 ميكروغرام / مل. من ناحية أخرىز أظهر الفحص الكيميائي النباتي الأولي عن وجود العديد من المكونات النباتية في مستخلصات النباتات المختبرة ، على وجه الخصوص ، التربينويدات ، الصابونين ، الفلافونويد والعفص. من جديد ، أظهرت كسور أسيتات الإيثيل التي تم الحصول عليها من G. alata و E. prostrata نتيجة ملحوظة من حيث محتوى المكونات النباتية الفينولية والفلافونويد والتانين. بالإشارة إلى نتائج النشاط المثبط لـ AChE في المختبر ، أظهر جزء أسيتات الإيثيل الذي تم الحصول عليه من G. alata أعلى نشاط مثبط لـ AChE (تثبيط 78 ٪ ، IC50 0.246 ± 0.02 مجم / مل) مقارنة بمستخلصات النباتات الأخرى والمعيار الأنشطة المثبطة للتحكم (إسيرين: 70٪ تثبيط ، IC50 0.00032 ± 0.01 مجم / مل). أشارت نتائج البيانات أيضًا إلى أن الكسر له معنوية (P <0.05) قيمة من مجموعة التحكم. من بين المكونات النباتية التسعة التي تم اكتشافها من GC / MS وعشرين مركبًا فينولًا تم اكتشف في النبات تم تثبيتها ضد الهياكل البلورية للأشعة السينية 4EY7 و 6WVC ، أظهر ثمانية عشر مركبًا درجات V أعلى من كليهما ، اللجينة المشتركة البلورية والتحكم الإيجابي القياسي مقابل عشرات. احتلت Quercetin المرتبة الأولى في قائمة الوضعيات (نقاط تصنيف LF). ومع ذلك ، من حيث التقاربات الملزمة ، تنبأت الحرائك الدوائية وملامح السمية مجتمعة ، كان الهيسبيدولين هو الأفضل (درجة Vs: -9.091 و -9.226 ، على التوالي). الخاتمـــــة : وجد أن جزء إيثيل أسيتات الذي تم الحصول عليه من G. alata أنه الجزء الواعد في سياق نشاط مضادات الأكسدة ، والنشاط المثبط لـ AChE في المختبر وفي برامج المحاكاة الحاسوبية. كما وجد أن مركب هيسبيدولين الفلافونويد المكتشف في نفس الجزء يحقيق أفضل ما يمكن توقعه من تثبيط الإنزيم ، وملامح الحركية الدوائية والسمية. تجدر الإشارة إلى أنه لم يتم العثور على دراسات سابقة في الأدبيات المتعلقة بنشاط تثبيط إنزيم AChE للنباتات الثلاثة قبل هذه الدراسة. تم اكتشاف العديد من المكونات النباتية في مستخلصات النباتات التي تم فحصها ومعظمها من المشتقات الفينولية. يقدم هذا العمل نظرة ثاقبة واعدة على أهمية إجراء مزيد من الدراسات على مستخلصات النباتات المدروسة تجاه عزل وتوصيف المكونات النباتية المثبطة لـ AChE. فيما يتعلق بالدراسة الحالية ، وجد أن مستخلصات أسيتات الإيثيل و البيوتانول التي تم الحصول عليها من النباتات الثلاثة المدروسة هي الأعلى نشاطا مضادا للأكسدة في المختبر ، ومن بين المستخلصات الخمسة عشر التي تم فحصها لنشاط تثبيط أسيتيل كولين استريز ، فقط جزء أسيتات الإيثيل من نبات القدقاد أظهر نشاط تثبيط معتبر لإنزيم الأسيتيل كولين استريز. وجد أن مركبات الفلافونويد والتانينات هي أكثر المكونات النباتية وفرة في جميع المستخلصات الخام الثلاثة. أظهرت الدراسة الحاسوبية أن مركب هيسبيدولين الموجود في نبات القدقاد هو الأقوى ارتباطاً ليكون أكثر المركبات وعوداً لتثبيط إنزيم أسيتيل كولينستراز البشري مع خصائص حركية دوائية جيدة.
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    In vitro Glycogen Phosphorylase Inhibition Coupled with LC/MS Profiling and Antioxidant Activity of Nauclea latifolia Smith Fruits
    (University of Khartoum, ) Abd Algaffar, Shereen Omer Elfarouk ; Sami Ahmed Khalid ; Pharmacognosy
    Background: The long-term effects of the current clinically used anti-diabetic drugs are still unsatisfactory with various adverse effects which limit their usage. Considering its central role in glycogen metabolism, glycogen phosphorylase (GP); an allosteric protein with at least six different ligand binding sites that modulate its enzymatic activity; has recently emerged as an important therapeutic target to discover potent antidiabetic drugs for type 2 diabetes (T2D). Meanwhile, recent evidence indicates the intricate association of the inflammatory damage characterizes diabetes and the pathophysiological events mediated through pancreatic islets reactive oxygen species (ROS) in the loss of β-cell function. Traditional medicine in Sudan have been used for centuries to treat diseases including diabetes, hence there are already vast literatures available for data mining and among them Nauclea latifolia fruit (Karmadoda) standing very prominently. Therefore, during the last few years, considerable attention has been given to scientifically validate the bioactivity of some antidiabetic traditional plants. The aim of this study is to validate the traditional use of N. latifolia fruit as antidiabetic agent and identify its chemical components, molecular target, and mechanism of action in comparison with the intact fruits. Methods: The plant including its fruits was taxonomically identified as N.latifolia Smith and herbarium specimens were deposited. Both the intact and processed fruits were extracted with 70% ethanol, concentrated, and fractionated with petroleum ether, chloroform and ethyl acetate respectively. The crude extract and all its respective fractions were screened for (GP) inhibition using GPa. Caffeine and quercetin were used as standards. The Antioxidant activity was performed according to standard methods using DPPH (2,2 Diphenylpicrylhydrazyl) and ABTS (2,2’- Azinobis( 3-benzothiazoline- 6 sulfonic acid). Scavenging capacity of the compounds was compared with that of Ascorbic acid and Trolox. The quantification of the flavonoids contents was executed according to Aluminum chloride colorimetric assay method and total flavonoid content was expressed as quercetin equivalent per 100g dry weight of sample. The total phenolic content was determined using Folin-Ciocalteu reagent and the results were expressed as gallic acid equivalents per 100g dry plant matter. LC-PDAESIMS analysis were performed on apparatus with an electrospray source assisted by the DataAnalysis® software. The online SciFinder Nauclea genus database “Nauclea DB” was used to predict from all NPs of Nauclea genus. SpectrusX processor, C+HNMR Predictors and DB software (ACD/Labs, 2014 Release) were used to predict the chemical shifts of these natural products. Molecular Operating Environment (MOE. v. 2008.10, Chemical Computing Group, Canada) and Discovery Studio Visualizer Softwares were used for the visualization of the docking results and generating the images. Results: Systematic screening of N.latifolia Smith intact fruit and its traditionally processed fruit (Karmadoda) revealed a remarkable GP inhibition for the intact fruit (95.57%) in comparison with the processed fruit. The DPPH and ABTS radical scavenging activity revealed that the intact fruit ethyl acetate fraction exhibited the most prominent antioxidant activity (89.50±.0..0%DPPH RSA± SD) which far exceeding its respective processed counterpart (83.57±0.02 %DPPH RSA± SD). Statistical analysis indicated that processing of fruits had a direct negative significant impact (P< 0.05) on both the antidiabetic and antioxidant activities. Quantification of both the total phenolics and total flavonoid contents revealed higher polyphenolic contents in the intact fruits compared to processed ones. Planar chromatographic analysis and hyphenated LC/MS techniques resulted in the identification of five β-carboline alkaloids, a flavonoid glycoside and chlorogenic acid in the intact fruit, while the processed fruit exhibited different chemical profiles characterised by the presence of only three β-carboline alkaloids besides the flavonoid glycoside in addition to a bis (2-ethylhexyl) phthalate which is considered as an artefact. Eventually, among the allosteric molecular targets analysed computationally, the catalytic site proved to be the most appropriate to accommodate both the phenolic and alkaloids identified in the fruit as ligands with the flavonoids luteolin 3'- methyl ether 7-glucuronide-4'-rhamnoside and chlorogenic acid scoring -28.39 and -21.42 Kcal/mol, respectively, among the phenolic compounds, as well as the 10- hydroxy strictosamide and strictosidine scoring - 28.40 and -23.07 Kcal /mol, respectively. Conclusions: The intact fruit is superior in terms of its antidiabetic and antioxidant capacity than the processed fruit. The catalytic site has been identified as the main N. latifolia molecular target for GP antidiabetic activity
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    Activity- guided Isolation and Structure Determination of Antioxidant and Antidiabetic Compounds from Bauhinia rufescence L.
    (University of Khartoum, ) Osman, Wadah Jamal Ahmed ; Abdelkhaleig Muddathir ; Pharmacognosy
    Background: Diabetes mellitus is a group of disorders of multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Diabetes remain one of the world’s major health problems and many repots have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. This study aimed to evaluate the antidiabetic and antioxidant activity of Bauhinia rufescens a plant used in Sudanese folkloric medicine for treatment of diabetes. It was also aimed to identify isolates and characterize the bioactive antidiabetic and antioxidant compounds using bioactivity guided fractionation followed by High Performance Thin Layer Chromatography (HPTLC) autobiography, Liquid-Mass spectroscopy analysis and nuclear magnetic resonance (NMR). Method: The powdered plant leaves were exclusively extracted using petroleum ether, chloroform and methanol, successively. These extracts were tested in streptozotocin-induced diabetic Wistar rats for antidiabetic as well as DPPH antioxidant potential. Standard Propyl Gallate was used as (+ve) control and the difference in results was considered significant when P < 0.05. The best active extract was fractionated with chloroform, ethyl acetate and butanol along with the remaining aqueous fraction. The fractions are tested for antioxidant and antidiabetic activity using HPTLC autobiography (α amylase and α glucosidase). Further, bioactive compounds were isolated by Preparative Thin Layer Chromatography and identified using LC/MS and NMR spectroscopy.X Results: The methanol extract significantly decreased the blood glucose level in normal fasted rats by 27 %; two hours following administration and decreased the blood glucose level in streptozotocin-treated rats by 48.9%; two hours following oral administration, while chloroform and pet. Ether extracts not significantly decreases the glucose level.Significantly the methanol extract also showed strong antioxidant activity (89%) compared with other extracts; thereforeit was further subjected for fractionation by using chloroform, ethyl acetate, n-butanol and water sequentially, and then tested for their antioxidant activity; where the ethyl acetate fraction was showed good activity compared with other fractions. Bioactivity guided fractionation of Bauhinia rufescensled to isolation and characterization of two compounds with potent antidiabetic and antioxidant activity namely; Quercetin-3-Orutinoside and Kaempferol -3-O-rutinoside. These compounds were isolated by using preparative thin layer c chromatographic techniques (PTLC) and their structures were identified by 1D NMR spectrum (C13and 1HNMR), 2D NMR (COSY, HSQC, HMBC Correlation) and confirmed bycomparing with reported data. LC/MS analysis led to identified more than thirty compounds; which were subjected to in silico assessment against human α-glucosidase and α-amylase enzymes. Insilico assessment showed that more than 14 compounds showed potent activity compared with acarbose (standard anti-diabetic drug). High performance thin layer chromatography (HPTLC) for the first time of the active fractionof the plant revealed the presence of different spots/peaks in different Rf values, corresponding to different compounds.XI Conclusion: This study validated the traditional uses of the evaluated plant in treating diabetes; beside that it was found a good antioxidant. The compounds which were isolated and identified may provide a new lead for more potent analogues for drug discovery. The anti-diabetic potential and antioxidant activity reported in present investigation is reported for the first time on the aerial plant. Further the isolated and characterized compounds from this study are also being reported for the first time. Toxicological studies may further be planned to develop it in the form of new drug for management of diabetes, however its use is available as per traditional claim.
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    Antimicrobial and Anti-inflammatory Activity of Plants Indiginous to Sudan
    (University of Khartoum, ) Ismaiel, Sarah Mustafa Osman ; Abd Elkhalig Muddather ; Pharmacognosy
    Atotal of 18 plant extracts belonging to 5 Sudanese medicinal plants, distributed among 5 families were screened for their antimicrobial and/or anti inflammatory activity. The plants are: 1. Acacia nilotica (L.)willd , F. Fabaceae (babul) 2. Hibiscus sabdariffa (L). ,F.Malvaceae (Roselle) 3. Hordeum vulgare(L)., F.Poaceae (Barley) 4. Salvadora persica (L). F.Salvadoraceae, (tooth brush tree) 5. Ziziphus spina-christi (L.)willd. ,F.Rhamnaceae (christs Thorn) For the antimicrobial activity ,the technique used was the cup plate agar diffusion method, using different solvents: chloroform, methanol, and water, they were tested against six microorganisms, four standard bacterial organisms one gram positive bacteria namely: (Staphylococcus aureus) and three gram negative bacteria (Eschrichia coli, , Proteus vulgaris and Pseudomonas aeruginosa) and two fungal organisms namely: (Aspergillus niger and Candida albicans) Out of the 18 extracts, 8 extracts (44.4%) exhibited inhibitory activity against one or more of the six tested microorganisms. Out ofthe 8 extracts showing antimicrobial inhibitory activity, 3(37.5%) were inhibitory to the six tested micro organisms. Eschrichia coli was the most susceptible microorganisms to the extracts, while Pseudomonas aerginosa showed the least susceptibility. The minimum inhibitory concentrations(MICs) of the most active extracts against standard organisms were determined. using agar dilution method. The antimicrobial activity of five reference drugs were determined against the six tested microorganisms and compared to the antimicrobial activity of the tested plant extracts. The most active extracs were four belonging to two plants :Acacia nilotica and Hibiscus sabdariffa in metahol and water ,then the MICs and the preliminary Phytochemical screening.was done. For the antiinflammatory activity , the method used was carrageenan induced oedema in albino rats. Using diclofenac sodium as reference anti inflammatory drug. The solvent was sodium carbonate. Out of 7 extracts 3 extracts (43%) exhibited antiinflammatory activity and decrease the oedema from the paw of the albino rats. The percent inhibition in the paw volume was determined for the 7 extracts after different interval and the data was estimated graphically. The most active extracts were three belonging to 3 plants. Acacia nilotica showed the highest antimicrobial activity , Hibiscus sabdariffa also revealed a good antimicrobial activity and Salvadora persica showed mild antimicrobial activity, MeOH/H2O extracts showed higher activity than chloroform ones, the other plants didn’t show any antimicrobial activity. Ziziphus spina-christi showed the highest anti inflammatory avtivity and Hordeum vulgare also showed mild antiinflammatory response while the other plants didn’t show any antiinflammatory activity. The preliminary Phytochemical screening of the 5 plants indicated that they contain many important chemical constituents including: saponins, tannins, sterols, triterpenoids, alkaloids, flavonoids, coumarins and cyanogenic glycosides.