Antioxidant and antibacterial constituents from Morus nigra

The objective of this study was to isolate and characterize the constituents of the local Morus nigra L. (Black mulberry), to compare its constituents with other studied Mulberries and to evaluate its antioxidative and anti-bacterial activities. The isolated compounds were identified by comparison of spectral data (UV, IR, MS and NMR) with literature values. The stem bark and wood of M. nigra yielded a stilbenoid oxyresveratrol 1, a 2-arylbenzofuran moracin M2, four isoprenylated flavonoids; cyclomorusin 3, morusin 4, kuwanon C5 and a derivative of kuwanon C6, two tritepenes; betulinic acid 7, -amyrin acetate 8 and a steroidal saponin -sitosterol-3-O-D-glucoside 9. The phenolic isolates showed moderate DPPH radical scavenging activity (EC50 = 23-135 μgml ) compared to ascorbic acid (EC50 = 41 μgml ) after 30 min. Compounds 1, 2 and 4 to 6 showed activities against Staphylococcus aureus, Bacillus subtilis, Micrococus flavus, Streptococcus faecalis, Salmonella abony, Pseudomonas aeruginosa.


INTRODUCTION
Morus nigra L. (Moraceae) belongs to the genus Morus which is widely distributed in Asia, Europe, North and South America and Africa.Mulberry (genus Morus) is an economically important plant used for sericulture, as a feed for the domesticated silkworm, Bombyx mori (Awasthi et al., 2004), and has a long history of medicinal use in Chinese medicine as a herbal medicine called "Sang Bai-Pi" (Nomura, 1988).The root bark, twigs and fruits which contain phenolic compounds are used as refreshing substances, are prescribed to treat cough, asthma, other chest complaints and rheumatism (Nomura, 1988).The decoction of the leaves possesses blood purifying properties, reduces fever and is diuretic (Kumar and Gupta, 1996).The bark of M. nigra was reputed to be used to expel tape worm and its extracts have been reported to have antibacterial and fungicidal activity.The chemical constituents of White Mulbery have been well studied by Taro Nomura (Nomura, 1988) who has reported phenolic glycosides, prenylated flavonoids, Diels-Alder type adducts, N-containing sugars, *Corresponding author.E-mail: mazimbaof@yahoo.comTel: +2673555273.Fax: +2673552836.
terpenoids, coumarins and 2-arylbenzofurans.The medicinal importance of the genus Morus prompted the phytochemical investigations of this plant.

Plant material
The dry stem wood of black mulberry (M.

General methods
Merck (Darmstadt, Germany) silica gel 60 (size 0.040 to 0.063 mm) was used for column chromatography.Thin layer chromatography (TLC) was carried out on 0.25 mm layer of Merck silica gel 60 F254 pre-coated on aluminium sheets.Merck silica gel 60 HF254 + 366 nm coated on 20 x 20 cm glass plates (0.5 mm thickness) were used for preparative TLC (PTLC).The UV light ( max 254 and 366 nm) and 1% vanillin-sulphuric acid spray were used for visualization.

DPPH radical scavenging assays
The antioxidant potential of M. nigra extracts and compounds was measured using the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) obtained from Aldrich (Munich, Germany) and following the method described by Yeboah and Majinda (2009).Briefly, methanolic samples (2 ml) of various concentrations (0.005, 0.05, 0.1, 0.2 and 0.5) mgml -1 were prepared and 2 ml of 2 % DPPH was added.Ascorbic acid was used as a standard and all measurements were done in triplicates.The absorbance of the mixture was measured at 517 nm at time intervals of 0.5, 1, 3 and 6 h.
The extracts of M. nigra fruit were reported to have a protective action against peroxidative damage to biomembranes and biomolecules (Naderi et al., 2004), while the roots methanolic extract showed mushroom tyrosinase inhibitory activity (Zheng et al., 2010).The flavonoids, 3-5 are C-3 and C-8 isoprenylated, and have closely related structures.Compounds 4 and 5 have higher free radical scavenging activities compared to 3 and 6, which have the C-3 isoprenyl group cyclised.This could be attributed to the involvement of the C 2 -C 3 double bond in the formation of partial aromaticity of the C 3 -C 2' pyrone rings.The higher activity of 4 over 5 was Each value is the mean ± standard deviation of a triplicate determinations, SD ± 0.04-, 0.08 µgmL -1 .SB = stem bark, SW = stem wood.attributed to the increased conjugation of 4 by cyclisation of C-8 prenyl group.The antibacterial activity of the extracts and compounds (1 to 6) was studied using the serial dilution method (Danielle, 2006) and tetracycline was used as a standard (Table 2).Compounds 1 and 2 showed bactericidal activity against S. aureus (MBCC = 125 and 62.5 µgml -1 resp.) and 2 also showed bactericidal activity against S. faecalis (MBCC = 500 µgml -1 ).The structure activity relationship of 3 and 4 showed that the cyclization of prenyl unit at C-3 in 3 reduced activity, whereas presence of free prenyl unit at C-3 in 4 enhances the activity.When comparing the activities of 4 and 6 it was observed that, when the prenyl unit at C-3 was cyclised (6) then to have better activity the C-8 attached prenyl unit should be open (6).Compound 3 has both prenyl units cyclised and 5 has both prenyl units open, these have lower activities than compounds 4 and 6 which have one of the prenyl units open.Though it is inconclusive, the results show that the electron donating group (-OH) at positions 7 and/or 2' was vital for increased activity as observed for 4 and 6.A more detailed study would have to be carried out to ascertain the role of hydroxyl groups, open and closed prenyl group in increased activity (Table 1).

Conclusion
The investigation of M. nigra bark and leaves yielded a stilbenoid, a 2-arylbenzofuran, four isoprenylated flavonoids, two tritepenes and a steroidal saponin.Moracin M (2) and Morusin, (4) were the only compounds to have been previously reported from M. nigra root and leaves, the other compounds have been reported from other Mulberries (M.alba, M. insgnis and M. macroura) except Black mulberry.The phenolic compounds showed moderate anti-oxidant and anti-bacterial properties.The results add to the use of phenolic compounds presence in Mulberries to partially explain their reported pharmacological activities which include use as refreshing substances and as antibacterial.
nigra) was collected from Gaborone, Botswana in June 2006.The stem bark was harvested from Selibe-Phikwe, in the Central District of Botswana in July 2006.The plant species was verified by Mr Andrew Muzila of the Herbarium, Department of Biological Sciences, University of Botswana.A voucher specimen (OM112) has been deposited in the University of Botswana Herbarium.

Table 1 .
DPPH radical scavenging activity of extracts and isolates from M. nigra

Table 2 .
Antibacterial activity of extracts and isolates from M. nigra