A Comparative Study of in Vitro Susceptibility of Madurella

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Original Article A comparative Study of In vitro Susceptibility of Madurella mycetomatis to Anogeissus leiocarpous Leaves, Roots and Stem Barks Extracts Ikram Mohamed Eltayeb*1, Abdel Khalig Muddathir2, Hiba Abdel Rahman Ali3 and Saad Mohamed Hussein Ayoub1

1Department of Pharmacognosy, Faculty of Pharmacy, University of Medical Sciences and Technology, P. O. Box 12810, Khartoum, Sudan 2Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan 3Commission of Biotechnology and Genetic Engineering, National Center for Research, Khartoum, Sudan

ABSTRACT

Objective: Anogeissus leiocarpus leaves, roots and stem bark are broadly utilized as a part of African traditional medicine against numerous pathogenic microorganisms for treating skin diseases and infections. Mycetoma disease is a fungal and/ or bacterial skin infection, mainly caused by filamentous Madurella mycetomatis fungus. The objective of this study is to investigate and compare the

antifungal activity of A. leiocarpus leaves, roots and stem bark

against the isolated mycetoma pathogen, M. mycetomatis fungus.

Methods: The alcoholic crude extracts, and their petroleum ether,

chloroform and ethyl acetate fractions of A. leiocarpus leaves, roots

and stem bark were prepared and their antifungal activity against the

isolated M. mycetomatis fungus were assayed according to the Address for NCCLS antifungal modified method and MTT assay compared to the Correspondence Ketoconazole, standard antifungal drug. The most bioactive fractions were subjected to chemical analysis using LC-MS/MS Department of chromatographic analytical method. Pharmacognosy, Results: The results demonstrated the potent antifungal activity of A. Faculty of Pharmacy, leiocarpus extracts against the isolated pathogenic M. mycetomatis University of Medical compared to the negative and positive controls. The chloroform Sciences and fractions showed higher antifungal activity among the other extracts, Technology, P. O. Box while the bark chloroform fraction was found to be the highest one. 12810, Khartoum, The chromatographic analysis of the chloroform fractions showed the Sudan presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, known for their antifungal activity and E-mail: kramela_07 toxicity to the filamentous fungi, steilbenoid compounds known as @yahoo.com phytoalexins secondary metabolites with potent antifungal activities

American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org Mohamed Eltayeb et al______ISSN 2321 – 2748

and the antimicrobial agents, flavonoids Conclusion: These studies present that the A. leiocarpus extracts posse’s potent antifungal activity against mycetoma causing pathogen compared to the ketoconazole standard drug and the highest activity was found to be in the stem bark of the plant.

Keywords: In vitro, Susceptibility, Madurella, mycetomatis, Anogeissus, leiocarpus, Leaves, Roots, Stem barks, extracts.

INTRODUCTION Combretaceae is a family of require amputation leading to loss of the flowering plants, widely distributed in the infected limbs33, 23, 34. tropical climates of Africa, Asia and South In Sudan mycetoma is a serious America. It incorporates 20 genera and common disease leading to the loss of around 600 species of shrubs, trees numerous limbs. The rate of mycetoma (evergreen or deciduous) or woody lianas1-3. infections in Sudan has not changed and The family is an important resource in around 400 new cases are found in clinics traditional medical practices for many and outpatient centers every year35, 33, 23, 34. human diseases3-8, many of these indications There is no potent and effective drug are related to treating infections7. The for treating mycetoma infection. efficacy of the plants may be due to the Ketoconazole is the favored antifungal presence of different classes of antimicrobial medication utilized for mycetoma secondary metabolites4, 5, 9. treatment36,23,34. Adequate treatment requires Anogeissus leiocarpus, is an African a prolonged antifungal drug combined with evergreen tree10 of genus Anogeissus extensive surgical treatment33, 23, 34. Meager belonging to this family with different uses data is available for susceptibility of M. in traditional medicine8,11-19. It mainly used mycetomatis to the plants secondary in treating skin diseases and infections, metabolites37. wounds infections, sore feet, boils, cysts, The present paper reported the syphilitic and diabetic ulcers13,20-23. The results of comparative study and activity plant was found to exhibited potent assessment of alcoholic leaves, barks and antibacterial and antifungal activity against roots extracts of the plant and their several pathogenic microorganisms24-30,19, chloroform fractions and ethyl acetate 31, 22, 32. fractions against M. mycetomatis. Emphasis Mycetoma is a chronic has been laid on the fungal susceptibility to granulomatous subcutaneous and deep the different metabolites occurring in tissues skin disease or a number of skin different morphological parts of the same infections caused by numerous fungi plant used in traditional therapy for the (eumycetoma) primarily Madurella infections treatment. mycetomatis fungus, or by bacteria (actinomycetoma). Progressive destruction EXPERIMENTAL of tissues leads to loss of function and impaired the affected site. Serious cases Plant Material The leaves, roots and stem barks of A. leiocarpus were collected from El

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Damazeine region in Sudan; their botanical hyphae which were harvested in identities were authenticated in the mycological peptone (BDH) water broth siliviculture department, Faculty of Forestry, medium with chloroamphenicol. The University of Khartoum, Sudan. The harvested mycelia or hyphae was washed voucher specimen was deposited at two to three times with RPMI 1640 with L- Department of Biochemistry, Commission glutamine medium, and then incubated for of Biotechnology and Genetic Engineering, 24 hours. The harvested mycelia, was National Centre for Research, Sudan. Barks sonicated for two minutes till homogenous and roots were chipped using saw mill, and suspension of mycelia was obtained. the plant materials were air dried under shade at room temperature, ground to a Anti Fungal Procedure coarse powder using electric grinder. While the leaves were ground into powder utilizing NCCLS Antifungal Modified Assay mortar and pestle. One ml of RPMI medium containing serially diluted extracts (10-0.31mg/ml) Preparations of the Extracts were placed in sterile test tubes, then 1ml of Plant powdered materials were prepared suspension was added. Tow set of extracted by maceration over night in 80% control tubes were used in the experiment, alcohol. Alcoholic extracts were fractionated one is growth control tubes(-ve) contained using solvents with gradually increasing 1ml of RPMI medium without any treatment polarities; petroleum, chloroform and ethyl and 1ml of prepared suspension, and the acetate). The obtained fractions were other one was standard drug (+ve) control concentrated by evaporation of the solvents tubes contained 1ml of RPMI medium with under reduced pressure using a rotary serially diluted ketoconazole (5-0.31mg/ml). vacuum evaporator. The optical density of the prepared growth control suspension was measured prior Madurella mycetomatis Collection incubation using a spectrophotometer at 680 M. mycetomatis Isolated fungus was nm red filter and reported as initial reading. collected from mycetoma research center at Then all test tubes were incubated at 37°C Soba hospital, Sudan. The black grains were for a week then, the optical density was exuded from open sinuses and surgical measured at 680 nm38, 39 biopsy from the lesion, freed from tissues MIC value is the least concentration and carried by forceps in sterile container before the spectrophotometer transmission with saline. reading is the same as or more than the initial reading37. It the least concentration, Culture and Preparation of Fungal when, there is no any growth of inoculated Suspension tested organisms had been seen40. The isolated grains were washed several time with saline solution and were MTT Assay firstly cultured in blood agar media,and then The assay is a quick sensitive sub--cultured in sabouraud dextrose agar colorimetric method; utilize tetrazolium salt and incubated at 37°C for 8 days. as indicator of microbial metabolism for The isolated strains were sub - evaluation of cell death41. This method is cultured again to maintain pure isolate of actually the reduction of yellow MTT salt hyphae. The subculture of hyphae was [tetrazolium salt (3-{4, 5-dimethylthiazole- repeated for two weeks to maintain pure 2-yl}-2, 5-diphenyl tetrazolium bromide)]

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into the green blue or violet blue formazan addition to the stem bark chloroform by the mitochondrial dehydrogenase, show fraction was found to be the most active just in the living cells and henceforth fraction. The results were compatible with discharged into the supernatant. The color the results of other related Anogeissus spp intensity is directly proportional to the living (Anogeissus latifolia) against skin disease cell numbers in the culture. One drop of the organisms44. indicator was added to all tested tubes after The leaves extract and fractions measuring the final optical density by a inhibited the inoculum initial reading 0.04 at spectrophotometer42,43. 680nm to 0.03, 0.02, 0.03, 0.02after a week inoculated in 10 mg/ml alcoholic extract, LC-Triple Quadruple Spectrometric chloroform, ethyl acetate and petroleum Analysis (LC-MS/MS) ether fractions respectively and to 0.03, 02, LC-MS/MS system was equipped 0.03, 0.03 when 5mg/ml is used. In with: comparison to the inocuulm growth reading HPLC column (RP-C18) and UV up to 0.23 in the negative control and detector (Diode array DAD) adjusted at 320 inoculum inhibition reading to 0.03 – 380 nm, coupled with Finnigan LCQ ion in5mg/ml ketoconazole positive control. trap mass spectrometer with the Chloroform fraction was found to be the Electrospray Ionization (ESI) interface at most potent. The results justified the negative ion mode for compounds traditional uses of the leaves decoction for detection. Collision induced dissociation treatment of skin diseases and infections. (CID) experiment was performed for The stem bark extract and fractions fragmentation of glycosides and elucidation showed higher activity than the leaves. The of compounds structures. initial inoculum optical density reading was inhibited to 0.02, 0.01, 0.03 when RESULTS AND DISCUSSIONS inoculated for a week in 10 mg/ml alcoholic extract, chloroform and ethyl acetate The results of antifungal NCCLS fractions consequently and to 0.02, 0.02, method are shown in figure 2 (a, b, c). The 0.03 in 5mg/ml. Chloroform fraction was optical density reading of the fungal found to be the most potent among the all suspension indicated the susceptibility of extracts and fractions of the three parts of fungus to the extracts. The susceptibility the plant, and it inhibited the inoculum was compared to the controls, one with reading up to 0.01 after a week inoculation ketoconazole standard drug (positive in 10mg/ml. It is noteworthy to add that control) and the other without drug (negative these findings were in agreement with the control). The optical density reading of the uses of stem bark decoctions in treatment of inoculum at 680nm was set at 0.04, as the skin diseases in African traditional initial reading. medicine. The results were compatible with The results showed that, all extracts the current literature of the stem bark and fractions inhibited the fungal growth extracts against skin disease caused by other with different degree. The extracts had organisms20. potential antifungal activity against M. The root extract and fractions mycetomatis contrasted with the showed less activity than the leaves and ketoconazole standard medication. The stem bark extracts and fractions. The chloroform fractions of the three parts of the inoculum initial reading was inhibited to plant showed higher activity than alcoholic 0.03, 0.04, 0.02 when inoculated for a week extracts and ethyl acetate fractions. In

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in 10 mg/ml alcoholic extract, ethyl acetate In the roots extracts the tetrazolium and chloroform fractions consequently and color started to change at the concentration to 0.03, 0.04, 0.03 in 5mg/ml. of 1.25mg/ml, 5mg/ml and 0.62mg/ml in These findings showed that, the alcoholic extract, ethyl acetate and extracts of the stem bark were more potent chloroform fractions, respectively. The than the leaves and roots extracts while the tetrazolium color changed at 0.31mg/ml in leaves extracts were more potent than the the ketoconazole drug. roots extracts. These results supported the The LC-MS/MS analysis of the traditional healer's use of the stem barks chloroform fractions with the higher activity more than leaves and root; the leaves more are shown in figure 3 (a, b, c) and table 1(a, than root in the treatment of skin disease45, b, c). 46, 20, 47, 48, 49, 27, 24. The LC-MS/MS analysis of the The MIC values of the extracts leaves chloroform fraction identified fifteen compared to the MIC of the control drug compounds of ellagic acid derivatives, (5mg/ml), was found to be 2.5mg/ml in flavonoids and steilbenoids. These findings alcoholic extracts of the three parts. The are reported for the first time with regard to MIC of the ethyl acetate and chloroform the reported results about the abundance of fractions of both bark and leaves were found ellagic, flavellagic acid derivatives and to be 5mg/ml and 0.62mg/ml respectively. flavonoids in the other member of the genus While in the root MIC of the chloroform Anogeissus52, 53, 44, 54, 55 . fraction was found to be 2.5mg/ml, and the Seven ellagic and flavellagic acids ethyl acetate fraction is active at10mg/ml. derivatives were identified in the chloroform The result was compatible with the MIC of stem bark fraction in agreement with antimicrobial agents reported by Banso et reported chemistry of this part of the plant56, al., 199950; Prescott et al., 200251 and Mann 57. et al., 2008b27. Nine compounds were identified in In the MMT results, the tetrazolium the root chloroform fraction. These results color changed represented the fungal viable are mentioned for the first time in the A. and growth. The results of the extracts leiocarpus root, in addition to the reported against the fungus in compared to the results about the abundance of ellagic, ketoconazole, standard antifungal drug flavellagic acid and flavonoids derivatives in showed that, the tetrazolium salt color in the other Anageissus species52, 44, 54. fungal suspension started to change at the The results of chromatographic concentration of 0.62mg/ml after a week analysis were compatible with the toxicity of inoculation in alcoholic leaves extract. In the ellagic acid against fillamentus fungi ethyl acetate, chloroform and petroleum presented by Scalbert,( 1991)58, in addition ether fractions the color started to change to that, the steilbenoid compounds were at 5mg/ml, 0.31mg/ml and 1.25mg/ml known as phytoalexins secondary respectively. metabolites with potent antifungal In the stem barks extracts the color activities59,60,61,62 and the flavonoid started to change at the concentration of antimicrobial agents63. 0.62mg/ml and 1.25mg/ml of alcoholic extract and ethyl acetate fraction CONCLUSIONS respectively. In the chloroform fraction there was no color change up to the concentration The M. mycetomatis fungus was of 0.31mg/ml. susceptible to A. leiocarpus extracts which showed potent antifungal activity against

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mycetoma causing pathogen compared to Spectra. Deepest appreciation to Mr. Ashraf the ketoconazole standard drug. None of the Hatium Osman, Lecturer at the University of extracts was found to enhance fungal Medical Science and Technology, Sudan, growth. Advanced hyphenated techniques for his valuable, kind helping and assistance. LC/DAD-MS/MS revealed the presence of The authors express many thanks and ellagic acid derivatives, stelbenoids and appreciation to the University of Medical flavonoids at different concentrations in the Science and Technology, Sudan, for aforesaid extracts. The ellagic acid providing support and encouragement to derivatives in the chloroform stem bark carry out the present work. fraction were found to be the highest in concentration, hence the highest toxicity REFERENCES against the M. mycetomatis filamentous fungus. The compounds in leaves 1. Watson L and Dallwitz MJ. The families of chloroform fraction with activity next to the flowering plants: descriptions, illustrations, identification, and information retrieval .1992; stem bark were found to be also ellagic acid http://delta-intkey.com/ (accessed 5 October derivatives but with less concentration than 2011). that in the chloroform stem bark fraction, in 2. Michael A. "Combretaceae" A dictionary of addition to antifungal stelbenoids Plant Sciences.1998; compounds. The moderate and least http://www.encyclopedia.com (accessed 5 concentrations of ellagic acid derivatives in October 2011). the leaves and root fractions enabled by the 3. Angeh JE, Huang X, Sattler I, Swan GE, antifungal stelbenoids to exert better activity Dahse H, Hartl A and Eloff JN.Antimicrobial followed by the antimicrobial favonoids in and anti-inflammatory activity of four known these fractions against the fungus. and one new triterpenoid from According to these findings which are based Combretumimberbe (Combretaceae). Journal of Ethnopharmacology 2007; 110(1): 56-60. on the results obtained it could be concluded 4. Eloff JN, Famakin JO and Katerere that, the activity against M. mycetomatis was DRP.Combretumwoodii (Combretaceae) leaf proportional to the concentration of ellagic extracts have high activity against Gram- acid derivatives, steilbenoid and flavonoids negative and Gram-positive bacteria. African in the extracts respectively. The ellagic acid Journal of Biotechnology 2005; 4 (10): 1161- derivatives were the most potent, followed 1166. by stelbinoids and finally the flavonoids. 5. Eloff JN, Famakin JO and Katerere DRP. Combretumwoodii (Combretaceae) leaf ACKNOWLEDGEMENTS extracts have high activity against Gram- negative and Gram-positive bacteria. African The authors are grateful to the Journal of Biotechnology 2005; 4 (10): 1161- Management and Staff members of the 1166. Mycetoma Research Center at Soba 6. Mann A, Amupitan JO, Oyewale AO, Hospital, Sudan for providing the M. Okogun JI and Ibrahim K. An ethnobotanical survey of indigenous flora for treating mycetomatis isolates. Many thanks to Mr. tuberculosis and other respiratory diseases in Elkhassein, University of Sciences and Niger State, Nigeria. J. Phytomed. Technology, Sudan, for his technical Therap 2007; 12: 1-12. encouragement to carry out this study. Our 7. Eloff JN, Katerere DR and McGaw LJ.The deepest thanks go to Prof. Nickoli Kuhnert biological activity and chemistry of the group, School of Engineering and Science, southern African Combretaceae. J. International University, Bremen, Germany Ethnopharmacol 2008; 119(3): 686-699. for assisting in running the Tandam Mass

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Table 1(a): RP-HPLC data (Peak NO. & Rt.), and MS/MS data (molecular weight {m/z} & main fractions {m/z}) and structure assignment of the leaves chloroform fraction

Compound M-H (R ) (min CID Mn Main Fraction ions (m/z) Compound Name Peak t (m/z) 425, 377, 301, 275, 271, 229, Di- hydroxyl, Tri-methoxy- 1 6.8 541 201,173 ellagic acid-7-O-β-glucoside Di- hydroxy, Tri-methoxy- 2 8.5 552 481, 301, 275, 271, 243 ellagic acid-7-O-β-glucoside derivative 459, 425, 377, 301, 275, 271, Di- hydroxyl, Tri-methoxy- 3 8.8 541 257, 227, 185,117 ellagic acid-7-O-β-glucoside 458, 436, 419, 401, 382, 351, 4 10.2 467 Ellagic acid-7-O-β-glucoside 313, 301, 275, 229 Di- hydroxy, Tri-methoxy- 601, 541, 522, 481,301, 299, 275, 5 12.4 617 ellagic acid-7-O-β-glucoside 271, 243 derivative Di- hydroxyl, Tri-methoxy- 623, 552, 481, 301, 275, 271, 6 12.4 628 ellagic acid-7-O-β-glucoside 243,187 derivative 312.7, 252.7, 222.7, 168.7, 7 12.7 453 E-Viniferin 168.7,150.7,124.8,124.8 453, 312.7, 252.7, 222.7,168.7, 8 12.7 490 Methyl E-Viniferin 168.7, 150.7.8 365, 300, 283, 271, 257, 243, Ellagic acid-4'-O-β- 9 15.7 447 229,170, 185,157,145,89 rhamnoside 463, 301, 300, 271, 255, 229, Quercetin-3-O-galloyl- 7-O- 10 15.7 615 193,178,151,107 β-glucoside 283, 271, 257, 240, 229, 228, 11 16.8 301 Ellagic acid 217, 202, 185, 173,139, 89 381, 301, 300, 271, 255, 229, Quercetin-7-O-β- 12 16.8 463 214,179,175, 151,107 glucopyranoside 365, 327, 285, 255, 227, 211, Kampefrol-7-O-β- 13 17.9 447 201,167,151,119 glucopyranoside 449, 360, 301, 285, 271, 255, Quercetin 3-methoxy-7-O- 14 18 477 243, 239, 211,123,179,163, β -glucopyranoside 151,107 365,301,300,283,271,255,229,21 Quercetin-7-O-β- 15 18.2 447 1,179,151,107 rhamnoside

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Table 1(b): RP-HPLC data (Peak NO. & Rt.), and MS/MS data (molecular weight {m/z} & main fractions {m/z}) and structure assignment of the barks chloroform fraction

Compound (R ) M-H CID Mn Main Fraction ions t Compound Name Peak (min) (m/z) (m/z) 2, 3, 8-Tri-methoxy- 506, 491,385, 342.8, 249, 16 6.8 521 flavellagic acid-7-β-O- 249, 155, 155, 155, 113 glucoside 3,4,3'-Tri-methoxy- 506, 385, 342.8, 249, 249, 17 20.2 521 flavellagic acid-4'-β-D- 155, 155, 155, 113 glucoside 248.6, 154.7, 152.7, 112.9, 18 20.2 384.8 Flavellagic acid derevative 112.9 Hydroxy, Tetra- methoxy - 19 21.6 359.0 343.8,32914,314,300, 285, ellagic acid 314, 299, 299, 285, 271, 3, 3'-Di-methoxy-ellagic 20 22.5 329 271 acid 328, 313, 313, 298, 298, 21 25.7 343 Tri-methoxy-ellagic acid 285, 270 Tetra-methoxy-ellagic acid 22 26.2 359 344, 329, 314,314,300, 285

Table 1(c): RP-HPLC data (Peak NO. & Rt.), and MS/MS data (molecular weight {m/z} & main fractions {m/z}) and structure assignment of the roots chloroform fraction

Compound (R ) M-H CID Mn main fraction t Expected compound Peak (min) (m/z) ions (m/z) 301, 275, 271, 229, Hydroxy, methoxy-ellagic acid 23 6.3 466 2001,185 -O- glucoside 312.7,252.7, 222.7, Hydroxy, Trimethoxy- flavone 168.7, 168.7, 150.7, 24 12.5 443 derevative 124.8, 124.8 257, 229, 200.7, 185.8, 25 14.3 274.8 Chalcone 172.7 300, 284, 257, 243, 229, 26 15.2 433 ElLagic acid- arabinoside 185, 172 327.7, 312.7, 312.7, Hydroxy, Trimethox- flavone 27 16.9 491 297.7, 297.7, 297.7, derevative 297.7, 284.7, 269.7 28 19.6 315 300, 272, 244, 216 Isorehamentein Tri-methoxy-flavellagicacid-O- 29 20.2 603 521, 506, 343, 328 β-glucoside derivative Methoxy- isorehamentein 30 21 613 329, 314, 314, 297, 271 derivative

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Di-hydroxy, dimethoxy- 31 22.4 328 299, 285, 271 flavones

Figure 1: Mycetoma pathogen collection

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

10mg 5mg 2.5mg 1.25mg 0.62mg 0.31mg 0.00mg

0.25

0.2

0.15

0.1

0.05 Optical transmission at nm 680

l l r e e o o e rm t l tr n th o ta o n a e f e z o th ro c a C e m o a n M u l l co le h y o o C th t tr E e e K P

Figure 2(a): In vitro susceptibility of M. mycetomatis to alcoholic extract, petroleum ether, chloroform and ethyl acetate fractions of A. leiocarpus leaves

10mg 5mg 2.5mg 1.25mg 0.62mg 0.31mg 0.00mg

0.25

0.2

0.15

0.1

0.05 Opticaltransmission 680at nm

0 l l o o m te le tr n r a o n a fo t z o h o ce a t r a n C e lo l o M h y c C h to t e E K

Figure 2(b): In vitro susceptibility of M. mycetomatis to alcoholic extract, chloroform and ethyl acetate fractions of A. leiocarpus stem barks

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

10mg 5mg 2.5mg 1.25mg 0.62mg 0.31mg 0.00mg

0.25

0.2

0.15

0.1

0.05 Optical transmission at 680 nm 0 l l o o m te le tr n r a o n a fo t z o h o ce a t r a n C e lo l o M h y c C h to t e E K

Figure 2(c): In vitro susceptibility of M. mycetomatis to alcoholic extract, chloroform and ethyl acetate fractions of A. leiocarpus roots

Intens. ANOLEAVESETOAC2.D: UV Chromatogram, 254 nm [mAU]

500

400

300

200

100

0 2 ANOLEAVESETOAC2.D: UV Chromatogram, 300-380 nm [mAU] 11 3000 5&6 10 12 14 2000 1 4 9 13 15 3 7 1000 8

ANOLEAVESETOAC2.D: UV Chromatogram, 280 nm [mAU] 300

200

100

0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min] Figure 3(a): RP-HPLC-DAD Chromatogram of A. leiocarpus leaves chloroform fraction recorded at λmax 254, 280,300-380nm

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Figure 3(b): RP-HPLC-DAD Chromatogram of A. leiocarpus barks chloroform fraction recorded at λmax 254, 280,300-380nm

Figure 3(c): RP-HPLC-DAD Chromatogram of A. leiocarpus roots chloroform fraction recorded at λmax 254, 280,300-380nm

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(541.2), 6.8min #294 x105 1.0 300.7

0.8 Compound 1: mz 541.8, 6.8mn 600.9 0.6

0.4 274.7 0.2 575.0 256.7 424.9 458.7 548.9 230.7 376.6 0.0 OH -MS3(541.2->300.6), 6.8min #296 3000 270.7 H3CO O OCH3 2500

2000 HO OH 1500

1000 228.7 172.6 200.7 256.7 283.7 O OCH3 500 O OH O 0 HO OH -MS4(541.2->300.6->270.8), 6.9min #298 120 198.6 100

80 OH OH 60 1,3,5,7 Tetrahydroxy 2,4,,8 trimethoxy ellagic acid-6-O-β-glucoside 40

0 150 200 250 300 350 400 450 500 550 600 m/z Figure: 4 (a) MS/MS (m/z) and assigned structure of compound (1) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(552.3), 8.5min #369 x105 622.9

1.5 Compound 2: mz 552.3, 8.5mn

1.0 300.8

481.0 0.5 274.7 322.7 570.9 596.9 0.0 x104 O -MS3(552.3->623.2), 8.5min #371 4

298.7 OCH 3 H3CO O 3 OH O OH 2 270.7 O

1 OH HO OCH 3 HO O 604.9 242.7 532.9 560.9 HO 504.9 0 O 6000 -MS4(552.3->623.2->298.9), 8.6min #373

5000 270.7 Di- hydroxy, trimethoxy-ellagic acid-7-O-β-glucoside derivative

4000

3000

2000

1000

0 150 200 250 300 350 400 450 500 550 600 650 m/z Intens. -MS2(541.1), 8.8min #382 x104 300.7

3 Compound 3: mz 541, 8.8 mn 600.9

2 458.7

1 274.7 574.9 376.6 424.9 530.9 248.7 316.7 398.7 505.0 633.1 0 x104 -MS3(541.3->300.6), 8.5min #370

270.7 1.0 OH

H CO OCH 0.8 3 O 3

0.6 226.6

HO OCH 0.4 3 256.7 0.2 184.6 116.9 O O OH 0.0 OH O HO OH

OH OH Di- hydroxy, trimethoxy-elagic acid-7-O-β-glucoside

100 200 300 400 500 600 m/z Figure: 4 (b) MS/MS (m/z) and assigned structures of compounds (2 &3) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(467.3), 10.2min #447 x105 274.7 1.25 Compound 4: mz 476, 10.2 mn

1.00 457.9 300.7 0.75 381.9 168.7 0.50

0.25 350.8 228.7 246.7 312.8 400.9 418.9 435.9 517.0532.9 200.7 0.00 x104 -MS3(467.3->274.6), 10.3min #449 1.25 O 228.7

1.00 HO O OH

0.75

O 0.50 HO O

0.25 256.7 OH OH HO O 156.7 202.7 0.00 HO O -MS4(467.3->274.6->229.2), 10.3min #451 1.5 Ellagic acid-7-O-β-glucoside

1.0

0.5

0.0

-0.5

-1.0 150 200 250 300 350 400 450 500 m/z Intens. -MS2(617.4), 12.4min #540 x105 Compound 5: mz 617, 12.4 mn 541.0 3

1 300.7 600.9

274.8 522.8 574.8 633.0 0 x104 OH -MS3(617.4->541.3), 12.4min #542

H3CO O 600.9 3 OCH3 300.7

2 HO OH

1 O OCH 274.7 O 3 574.9 OH O 424.9 549.0 248.7 392.9 HO 450.9 519.0 0 OH -MS4(617.4->541.3->601.1), 12.5min #544 3000

2500 298.7 OH 2000 OH

1500 270.7 1,3,5,7 Tetrahydroxy 2,4,,8 trimethoxy ellagic acid-6-O-β-glucoside 1000

242.6 500 214.7 528.0 0 200 300 400 500 600 m/z Figure: 4 (c) MS/MS (m/z) and assigned structures of compounds (4 & 5) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(628.5), 12.4min #541 x104 4 Compound 6: mz 628, 12.4 mn 622.9

3 300.7 552.0 2

481.0 1 OH 274.7 570.9 596.9 228.8 658.9682.9 0 H3CO O OCH3 -MS3(628.5->623.2), 12.4min #543 5000 298.7

4000 HO OCH3

3000

O OH 2000 O OH O 270.7 HO 560.8 1000 OH 605.0 242.7 532.8 186.6 340.7 505.9 579.0 0 -MS4(628.5->623.2->298.8), 12.5min #545 150 OH 125 270.7 OH

100 1,4,5,7 Tetrahydroxy 2,3,8 Trimethoxy ellagic acid-6-O-β-glucoside

0 100 200 300 400 500 600 m/z Intens. -MS2(453.3), 12.7min #553 x106

1.25 312.7 Compound 7: mz 453, 12.7 mn 1.00

0.75

0.50 168.7

0.25 182.7 224.8 252.8 288.8 0.00 x105 -MS3(453.3->312.9), 12.7min #555

2.0 168.7

1.5 HO

1.0 O OH

0.5 124.8

106.9 150.7 192.7 222.6 252.6 0.0 -MS4(453.3->312.9->168.7), 12.7min #557 HO 2500 124.8 OH 2000 -Viniferin

1500 OH

1000

500

0 100 150 200 250 300 350 400 450 m/z

Figure: 4 (d) MS/MS (m/z) and assigned structures of compounds (6 & 7) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(446.9), 15.7min #688 x105 3.0 299.7 2.5

2.0 Compound 9: mz 446.9, 15.7mn

1.5

1.0

0.5 364.8 282.7 0.0 -MS3(446.9->300.1), 15.8min #690

3000 243.6

256.7

2000

184.7 O 1000 270.7 199.6 HO O 89.0 144.7 228.7 283.7 156.8 0 -MS4(446.8->300.7->256.7), 15.5min #678 200 O O OH

184.7 H3C OH 150 OH HO HO O 228.6 100 O Ellagic acid- rhamnoside 50

0 100 150 200 250 300 350 400 450 500 m/z

Figure: 4 (e) MS/MS (m/z) and assigned structures of compounds (8 & 9) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(615.3), 15.7min #687 x106 1.5 463.0

Compound 10: mz 615.3, 15.7mn 1.0

300.8

0.5

270.7 0.0 x105 -MS3(615.3->463.2), 15.7min #689 OH 300.7 6 OH

O 4 HO O O 299.8 OH

2 HO HO O

150.7 254.8 O 0 OH O x104 -MS4(615.3->463.2->300.6), 15.8min #691

HO 270.7 3 150.7 178.7 HO OH 2 254.7 Quercetin 3-O-galloyl-7-O-glucoside

106.9 192.7 228.7 0 100 200 300 400 500 600 m/z

Intens. -MS2(300.8), 16.8min #739 8000 256.7

Compound 11: mz 300.8, 16.8mn 6000

4000

228.7 184.7 216.7 270.7 2000 200.7 283.7 138.8 239.6 89.0 172.6 0 -MS3(300.8->256.8), 16.9min #741

201.6 250 O

200 HO O

227.6 150 HO OH

100

O OH 50 O Ellagic acid

0 100 150 200 250 300 350 400 m/z

Figure: 4 (f) MS/MS (m/z) and assigned structures of compounds (10 & 11) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(463.1), 16.8min #738 x105

300.7 4 Compound 12: mz 463.1, 16.8mn 3

299.8 2

1 270.7 150.7 178.7 254.7 380.7 0 x104 -MS3(463.1->300.4), 16.8min #740 5 270.7 4 178.7

150.7 3

2 254.7

106.9 192.7 228.7 OH 0 -MS4(463.1->300.4->271.1), 16.9min #742 1000 OH

242.6 O 800 HO O O 228.7 OH 600 OH HO HO 400 213.6 OH O 198.6 254.6 200 174.5 Quercetin 7-0--glucopyranoside

0 100 150 200 250 300 350 400 450 500 m/z

Intens. -MS2(447.1), 17.9min #791 x105

2.5 283.8

2.0 Compound 13: mz 447.1, 17.9 mn

1.5 254.7 1.0

0.5 364.7 226.7 326.8 150.7 0.0 x105 -MS3(447.1->284.3), 18.0min #793

1.25 254.7 OH O 1.00 HO O O 0.75

0.50 HO OH HO HO 0.25 226.7 OH O 200.7 0.00 -MS4(447.1->284.3->255.0), 18.0min #795 1250 226.7 Kampefrol-7-O-β-glucopyranoside 1000

750

210.8 500 166.8

250 118.8

0 100 150 200 250 300 350 400 450 m/z

Figure: 4 (g) MS/MS (m/z) and assigned structures of compounds (12 & 13) in the chloroform fraction of A. leiocarpus leaves

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(477.2), 18.0min #792 x105

300.8313.8 1.5 Compound 14: mz 477.1, 18.0 mn

1.0

0.5 284.8 242.8 150.8 356.9 449.0 0.0 -MS3(477.2->300.6), 18.0min #794 6000 150.7 5000 270.7 4000 178.7

3000 254.6 2000

1000 106.9 238.7 162.6 192.7 210.7 285.8 0 OH -MS4(477.2->300.6->150.8), 18.1min #796 1.5 OH

1.0 O HO O O

0.5 OH OCH HOHO 3 0.0 OH O

-0.5 Quercetin 3-methoxy-7-O- β-glucopyranoside

-1.0 100 150 200 250 300 350 400 450 500 m/z

Intens. -MS2(446.9), 18.2min #805 x105

2.0 Compound 15: mz 446.9, 18.2 mn 300.7

1.5

1.0 299.8

0.5 282.7 364.7 178.7 0.0 x104 -MS3(446.9->300.6), 18.3min #807

1.25 178.6

150.7 1.00 270.7 0.75

0.50 254.7

0.25 228.7 106.9 210.7 0.00 -MS4(446.9->300.6->178.7), 18.3min #809 OH 50 150.8 O 40 O O OH OH

30 H3C OH 20 HO HO OH O 10 Quercetin-7-O- rhamnoside

0 100 150 200 250 300 350 400 450 500 m/z

Figure: 4 (h) MS/MS (m/z) and assigned structures of compound (14 &15) in the chloroform fraction of A. leiocarpus leaves

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Intens. -MS2(520.9), 6.8min #317 5 x10 Compound 16: mz 520.9, 16.8 mn 248.6 384.7 3

154.6 426.7

0 x105 -MS3(520.9->384.8), 6.9min #319

2.0 248.6 O 1.5

H 3CO O OCH 3 1.0

0.5 H O O O OCH 3 152.7 0.0 x104 OH -MS4(520.9->384.8->248.6), 6.9min #321 OH 4 H O O 154.7 H O

3 O 2, 3, 8-trimethoxy-flavellagic acid-7-O-β-glucoside 2

112.8 0 100 200 300 400 500 600 m/z

Intens. -MS2(521.0), 20.2min #934 x105 6 Compound 17: mz 521.o, 20.2 mn 384.8 248.7

342.8 506.0 2 490.9 426.8 154.7 0 x105 -MS3(521.0->384.8), 20.3min #936

3 248.6

O 2

H 3CO O OH

1 HO O 154.7 O OCH 3 0 x104 -MS4(521.0->384.8->248.6), 20.3min #938 OH 6 OCH H O O 3 154.6 HO

4 O

3,4,3'-tri-methoxy-flavellagicacid-4'- β-D-glucoside 2

112.9

0 100 150 200 250 300 350 400 450 500 550 m/z

Figure: 4 (i) MS/MS (m/z) and assigned structures of compounds (16 & 17) in the chloroform fraction of A. leiocarpus stem barks

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(384.8), 20.2min #935 x106 248.6 0.8 Compound 18: mz 384.8, 20.2 mn

0.6

0.4

0.2 154.7 0.0 x105 -MS3(384.8->248.6), 20.3min #937

152.7 1.5

O 1.0 H O O OH

0.5

112.9 OH 0.0 x104 -MS4(384.8->248.6->153.6), 20.3min #939

1.5 112.9 O OH

1.0 O

Flavellagicacid-derevative 0.5

180.6

59.5 0.0 50 100 150 200 250 300 350 400 450 m/z

Intens. -MS2(359.0), 21.6min #1004 x106 1.5 Compound 19: mz 359.0, 21.6 mn 343.8

1.0

328.8 0.5

0.0 x106 -MS3(359.0->344.0), 21.7min #1006 O

1.0 328.7 H 3CO O 0.8

0.6 H 3CO OCH 3 0.4

0.2 313.8 284.7 300.7 O OCH 3 0.0 H O

Hydroxy,tetra, methoxy - ellagic acid

100 150 200 250 300 350 400 450 m/z

Figure 4 (j) MS/MS (m/z) and assigned structures of compounds (`18 & 19) in the chloroform fraction of A. leiocarpus stem barks

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(329.0), 22.5min #1046 x106

Compound 20: mz 329.0, 22.5 mn 313.7 0.8

0.6

0.4 O 0.2

HO O 298.8 0.0 x105 -MS3(329.0->313.9), 22.5min #1048 4

298.7 3 HO OCH 3

2 O OCH 3 1 O 270.7284.7 0 x104 -MS4(329.0->313.9->298.8), 22.6min #1050 8 3, 3'-di- Di- methoxy - ellagic acid

270.7 6

0 100 150 200 250 300 350 400 450 m/z

Intens. -MS2(343.0), 25.7min #1211 x106 1.0 Compound 21: mz 343.0, 25.7 mn 327.8

0.8

0.6

0.4

0.2 O 312.7 0.0 H3CO O x105 -MS3(343.0->327.9), 25.7min #1213

312.7 3 HO OCH 3

OCH 1 O 3 284.7 O 297.8 0 x104 -MS4(343.0->327.9->312.8), 25.8min #1215 8 Tri- methoxy - ellagic acid 297.7

2 284.7

256.6 269.7 0 200 220 240 260 280 300 320 340 360 380 m/z

Figure: 4 (k) MS/MS (m/z) and assigned structures of compounds (20 & 21) in the chloroform fraction of A. leiocarpus stem barks

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

Intens. -MS2(359.0), 26.2min #1238 x106 3 343.8 Compound 22: mz 359.0, 26.2 mn 2

328.8

0 O x106 -MS3(359.0->344.0), 26.3min #1240 H C O O 3 328.7 2.0

1.5 H O OCH 3 1.0

0.5 O OCH 3 313.7 0.0 x106 H C O -MS4(359.0->344.0->328.9), 26.3min #1242 1.0 3

Hydroxy-tetra- methoxy- ellagic acid 313.7 0.8

0.6

0.4

0.2 300.7

285.7 0.0 225 250 275 300 325 350 375 400 425 m/z

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 6.3min #951, [mAU] 200 Compound 23: mz 466.3, 6.3 mn 100

0 Intens. -MS2(466.3), 6.3min #277 5 x10 300.7

2 OH 274.8 456.9 1 424.9 248.7 HO O 492.9 340.8 375.9 534.0OH 569.0 630.9 0 Intens. -MS3(466.3->300.7), 6.4min #279 270.7 3000 O 200.6 HO O 2000 228.7 1000 184.7 156.8 OCH 3 0 OH O Intens. HO -MS4(466.3->300.7->270.8), 6.4min #281 300 HO 242.8 H O 200 Hydroxy methoxyellagic acid -O- glucoside 100

0 200 300 400 500 600 m/z

Figure: 4 (l) MS/MS (m/z) and assigned structures of compounds (22) in chloroform fraction of stem bark and (23) in the root of A. leiocarpus

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

200 220 240 260 280 300 320 340 360 380Wavelength [nm] Intens. UV, 12.5min #1883, [mAU] 40 Compound 24: mz 443.2, 12.5 mn 20 0

Intens. -MS2(443.2), 12.5min #546 5 x10 312.7 4 168.7

2 124.9 150.7 222.7 252.7 OCH 0 3 Intens. -MS3(443.2->312.7), 12.6min #547 4 x10 168.7 OCH 3 6 4 H 3 CO O 2 124.8 150.7 222.7240.7 0 Intens. -MS4(443.2->312.8->168.7), 12.6min #548 HO 2000 124.7 O 1000 Hydroxyl trimethoxy flavone derevative 0 100 150 200 250 300 350 400 450 500 550m/z

200 220 240 260 280 300 320 340 360 380Wavelength [nm] Intens. UV, 14.3min #2147, [mAU]

100 Compound 25: mz 274.8, 14.3 mn 50 0 Intens. -MS2(274.8), 14.3min #624 4 OH x10 256.6 6 4 2 228.7 H O OH 202.7 218.7 244.6 0 Intens. -MS3(274.8->256.6), 14.3min #626 6000 228.6

4000

2000 116.8 172.7 185.8 200.7 Intens.0 OH OH -MS4(274.8->256.9->228.7), 14.4min #628 60 Hydrochalcone 40 20 0 100 150 200 250 300 m/z

Figure: 4 (m) MS/MS (m/z) and assigned structures of compounds (24 & 25) in the chloroform fraction of A. leiocarpus roots

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 15.2min #2289, [mAU] Compound 26: mz 433.2, 15.2 mn 25 0 O -25 Intens. -MS2(433.2), 15.2min #665 5 HO O x10 299.7 4

2 O O OH 0 Intens. OH -MS3(433.2->299.7), 15.3min #667 256.7 4000 OH 228.6 HO HO O 2000 184.8 200.7 283.7 O 171.7 242.5 0 Intens. -MS4(433.2->300.3->256.7), 15.3min #669

1 Ellagic acid -7-O-β- arabinoside

-1 150 200 250 300 350 400 450 500 m/z

200 220 240 260 280 300 320 340 360 380Wavelength [nm] Intens. UV, 16.9min #2540, [mAU] Compound 27: mz 491.2, 16.9 mn 50

0 OCH 3 -50 Intens. OCH 3 -MS2(491.2), 16.9min #740 6 x10 327.7 H 3 CO 0.50 O 312.7 0.25 475.9 297.7 0.00 Intens. HO -MS3(491.2->327.7), 17.0min #742 6 x10 312.7 1.0 O Hydroxyl trimethoxy flavone 0.5 derevative 297.7 0.0 Intens. -MS4(491.2->328.0->312.7), 17.0min #744 x105 3 297.7 2 1 284.7 269.7 0 150 200 250 300 350 400 450 500 550 m/z

Figure: 4 (n) MS/MS (m/z) and assigned structures of compounds (26 & 27) in the chloroform fraction of A. leiocarpus roots

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 19.6min #2942, [mAU] Compound 28: mz 315.0, 19.6 mn 0 OH

-100 OH

Intens. -MS2(315.0), 19.6min #862 5 O x10 HO 299.7

1.5 OCH 3 1.0 OH O 0.5 Isorhamentein 0.0 Intens. -MS3(315.0->299.7), 19.6min #864

800 243.6

600

400 215.7 200 271.6 0 150 200 250 300 350 400 m/z

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 20.2min #3028, [mAU] Compound 29: mz 603.3, 20.2 mn 0 -100 -200 Intens. -MS2(603.3), 20.2min #889 5 x10 521.0

1 O 0 Intens. -MS3(603.3->521.0), 20.2min #891 4 x10 342.8 H3CO O OH 4 506.0

2 HO O O OCH 3 0 Intens. -MS4(603.3->521.2->343.0), 20.3min #893 4000 OH 327.7 OCH HO O 3 HO 2000 O Tri-O-methylflavellagic acid-7-O-β-glucoside derivative 0 100 200 300 400 500 600 700 800 900 m/z

Figure: 4 (o) MS/MS (m/z) and assigned structures of compounds (28 & 29) in the chloroform fraction of A. leiocarpus roots

AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al______ISSN 2321 – 2748

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 21.0min #3155, [mAU] Compound 30: mz 613.3, 21.0 mn 0

-200 Intens. -MS2(613.3), 21.0min #929 6 x10 328.8

1 313.8

OCH 3 270.7 428.9446.9 0 Intens. OH -MS3(613.3->328.8), 21.0min #930 6 x10 313.7 0.75 O HO 0.50 0.25 OCH 3 0.00 Intens. OH O -MS4(613.3->328.9->313.8), 21.1min #931 5 x10 Isorham netin 3- m ethoxy derivative 3 298.6 2 1 270.7 0 250 300 350 400 450 500 550 600 650 m/z

200 220 240 260 280 300 320 340 360 Wavelength [nm] Intens. UV, 22.4min #3361, [mAU] Compound 31: mz 327.8, 22.4 mn 0

-200

Intens. -MS2(327.8), 22.4min #994 6 OCH 3 x10 313.7 1.0 OH

0.5 O HO 0.0 Intens. -MS3(328.9->313.7), 22.4min #996 x105 OCH 3 298.7 4 OH O 2 Iso rh a m n e tin 3 - m eth o xy 284.7 0 Intens. -MS4(328.9->313.9->298.7), 22.5min #998 x104 6 270.7 4 2 0 150 200 250 300 350 400 450 m/z

Figure: 4 (p) MS/MS (m/z) and assigned structures of compounds (30 & 31) in the chloroform fraction of A. leiocarpus roots

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