African Journal of Biotechnology Volume 15 Number 14, 6 April 2016 ISSN 1684-5315
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African Journal of Biotechnology
Table of Content: Volume 15 Number 14 6 April, 2016
ARTICLES
In vitro antimicrobial properties of friedelan-3-one from Pterocarpus santalinoides L’Herit, ex Dc 531 Ichiko Chic Odeh, Terrumun Amom Tor-Anyiin, John Ogbaji Igoli and John Vershima Anyam
Nanoformulation and antibiotic releasing property of cefotaxime nanoparticles 539 Maysaa Chasib Al-Moahmmedawi
Polymorphism of growth hormone gene and its association with wool traits in Egyptian sheep breeds 549 Ibrahim M. Farag, Ahmed M. Darwish, Hassan R. Darwish,
AbdelAziz K. B., Ramadan W. A., Mohamed M.I. and Othman E. Othman
Effects of diets containing Cissus rotundifolia flour on lipid profile of rats and postprandial glucose levels of normoglycemic human adults 557 Uchenna Agatha Onyechi and Vivienne Nkiruka Ibeanu
Table of Content: Volume 15 Number 11 16 March, 2016
Vol. 15(14), pp. 531- 538, 6 April, 2016 DOI: 10.5897/AJB2015.15091 Article Number: 8F932F357890 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
In vitro antimicrobial properties of friedelan-3-one from Pterocarpus santalinoides L’Herit, ex Dc
Ichiko Chic Odeh1, Terrumun Amom Tor-Anyiin2*, John Ogbaji Igoli2 and John Vershima Anyam2
1College of Education, Oju, Benue State, Nigeria. 2Department of Chemistry, Federal University of Agriculture, Makurdi, Nigeria.
Received 8 November, 2015; Accepted 25 February, 2016
Chemical investigation of ethyl acetate leaf-extract of Pterocarpus santalinoides led to isolation of a friedelane-terpenoid. Structure elucidation was done by comparison of the Nuclear Magnetic Resonance (NMR) spectral features with literature data and was consistent with reported values for friedelan-3-one. The following microorganisms (Clinical isolates): methicillin resistant Staphylococcus aureus (MRSA), S. aureus, Corynebacterium ulcerans, Streptococcus pneumoniae, Campylobacter jejuni, Helicobacter pylori, Escherichia coli, Shigella dysenteriae, Candida tropicalis and Candida krusei were used for the in vitro antimicrobial activity. The isolated compound had Minimum Inhibition Concentration (MIC) value of 10 µg/ml for MRSA, H. pylori, E. coli and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) of 40 µg/ml for E. coli; 20 µg/ml for MRSA, H. pylori and C. krusei; 10 µg/ml for S. aureus, S. pneumonia and C. tropicalis. It showed moderate antimicrobial activity against most of the pathogens tested. This is also the first report of the isolation of friedelan-3-one from this species.
Key words: Friedelan-3-one, methicillin resistant Staphylococcus aureus (MRSA), Helicobacter pylori, Escherichia coli.
INTRODUCTION
Plant based traditional medicine systems have continued and antimicrobial activities of its leaf-extracts have been to play an essential role in the health care of many reported (Odeh and Tor-Anyiin, 2014). cultures. Medicinal plants have remained vital in man’s The plant P. santalinoides (L’Herit ex Dc), family fight against disease (Phillipson, 2001). Pterocarpus Papilionoideae is a tree that grows along riverine forests santalinoides is a plant used by Igede people of North- of Africa and tropical South America and is a native of Central Nigeria for its antidiarrheal properties (Nworu et Brazil, Cameroon, Ghana, Nigeria, and Senegal al., 2009; Odeh and Tor-Anyiin, 2014). Phytochemical (Osuagwu and Akomas, 2013). P. santalinoides is
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 532 Afr. J. Biotechnol.
monoecious, flowering from December to March, fruit Antimicrobial activity of PS vlc-1 ripening between March and April. The Nigerian species are trees with light yellow flowers and they usually have Cultivation and standardization of test organisms alternate leaflets (Keay, 1989; Agroforestry Tree Tests were carried out using the following microorganisms (Clinical Database, 2011; Osuagwu and Akomas, 2013). isolates): methicillin resistant Staphylococcus aureus (MRSA), S. Extracts from this plant have been utilized for their aureus, Corynebacterium ulcerans, Streptococcus pneumoniae, antifungal and antibacterial activities among the Igede Campylobacter jejuni, Helicobacter pylori, Escherichia coli, Shigella people of Benue State, Nigeria in treatment of dysenteriae, Candida tropicalis, and Candida krusei. The test microbes were obtained from the Department of Medical inflammation of lower abdomen, stomach ache, and other Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, infectious diseases (Igoli et al., 2003). In this study, Nigeria. Pathogens were checked for purity and maintained in isolation, characterization, and anti-microbial properties slants of Agar. of friedelan-3-one from the leaf-extracts of the plant are Test organisms (loop full) were taken from agar slants and sub reported. cultured into test tubes containing Sterile Nutrient Agar (Oxoid Limited, England) for bacteria and Sabouraud Dextrose Agar (Titan Biotech Limited, Rajasthan, India) (20 ml) for fungi. Test tubes were incubated for 48 h at 37°C. The broth culture was standardized MATERIALS AND METHODS using Sterile Normal Saline (prepared from NaCl, bought from Sigma-Aldrich Chemical Company Inc, USA) to obtain a density of Plant 106 CFU/ml for bacteria. A sporulated test fungal spore was harvested with 0.05% TWEEN® 80 in Sterile Normal Saline and Leaves of P. santalinoides L'Hér. ex DC. Papilionoideae were standardized to 106 spores/ml. collected in March, 2014 from Anyuwogbu-Ibilla, Oju L.G.A, Benue State, Nigeria. The plant was identified and authenticated by Dr. S. A. Shomkegh of Department of Social Forestry and Environmental Antimicrobial profile Protection, University of Agriculture, Makurdi; a voucher specimen was also deposited. All solvents reagents used for this work were ® PS vlc-1 (0.004 mg) was weighed and dissolved in DMSO (Sigma- purchased from Sigma-Aldrich with the exception of Silica gel 60 Aldrich Chemical Company Inc., USA) (10 ml) bringing it to a F254 Thin Layer Chromatography Plates and Silica gel G which were ® concentration of 40 µg/ml in preparation for antimicrobial assay. All purchased from Merck KGaA, Germany. Solvents though of media were prepared according to manufacturer instruction, analytical grade were redistilled to remove possible traces of sterilized at 121°C for 15 min and were poured into sterile petri plastic. dishes, allowed to cool, and solidify. Disc diffusion method was used for screening of the extract. Sterilized media were seeded with standard inocula (0.1 ml) of test microbes, Mueller-Hinton Agar Preparation of extracts (Oxoid Limited, England) for the bacteria and Sabourand Dextrose Agar for the fungi. Inocula were evenly spread over the surface of The leaves were air-dried at room temperature in the chemistry media using sterile swabs. For each inoculated medium, a well (6 laboratory, University of Agriculture, Makurdi. The powder was mm) was cut at the centre using a standard cork-borer (6 mm obtained using mortar and pestle. The powdered plant material (1 diameter). kg) was extracted with hexane (2.5 L), ethyl acetate (2.5 L) and Agar diffusion method was used for antibacterial screening. methanol (2.5 L) for 8 h successively using a Soxhlet apparatus. Solutions of PS vlc-1 (0.1 ml) were introduced into each well of The extracts were concentrated and allowed to dry in a fume hood inoculated media. Inoculated media were incubated at 37°C for 24 to give the crude extracts, and were coded: PSH, PSE, and PSM h for bacteria and at 30°C for 7 days for the fungi, after which plates for hexane, ethyl acetate, and methanol, respectively. Their yields were observed for zones of inhibition of growth. Diameter of zone of were recorded. inhibition was measured with a transparent ruler and the result was recorded in millimetres.
Phytochemical screening Determination of minimum inhibitory concentration (MIC) The crude extracts were subjected to various phytochemical tests to identify the chemical constituents (Sofowora, 1982; Hassan et al., MIC was carried out using broth dilution method. Mueller Hinton 2004; Edeoga et al., 2005; Anowi et al., 2012a; Anowi et al., and Sabouraud Dextrose broth were prepared according to the 2012b). manufacturer’s instruction. The broth (10 ml) was dispensed into test tubes, separated, and was sterilized at 121°C for 15 min and allowed to cool. Mc-Farland’s turbidity standard scale number of 0.5 Fractionation and isolation was prepared to give a turbid solution. Normal saline was prepared and was used to make a turbid suspension of the microbes. The The ethyl acetate extract was pre-adsorbed onto Celite prior to Normal Saline (10 ml) was dispensed into test tubes and test purification by Vacuum Liquid Chromatography (VLC) on silica gel. microbes were inoculated and incubated for 6 h at 37°C. Dilution of The column was eluted with gradient mixtures of hexane-ethyl micro-organisms in Normal Saline was continuously done until the acetate, starting from [100% hexane: 0% ethyl acetate] to [0% turbidity (1.5×106 CFU/ml) matched that of the Mc-Farland scale by hexane: 100% ethyl acetate] and ethyl acetate-methanol ranging visual comparison. Two-fold serial dilution of extract in sterile broth from [100% ethyl acetate: 0% methanol] to [0% ethyl acetate: 100% gave concentrations of 20, 10, 5, and 2.5 µg/ml. Having obtained methanol] as previously described by Tor-Anyiin et al. (2015). different concentrations of extracts in the broth, a standard Eluents were analyzed by thin layer chromatography (TLC). inoculum (0.1 ml) of the microbes was inoculated into the different Fraction PS vlc-1 was obtained as a white solid [with melting point concentrations. Incubation for bacteria was at 37°C for 24 h and at (260 to 265°C), Rf × 100 (76)]. 30°C for one week for fungi. Test tubes were then observed for Odeh et al. 533
29 30
Figure 1 1. . StructureStructure of of friedelan friedelan-3--one3-one isolated isolated from from leaves leaves of of Pterocarpus santalinoi santalinoides.des.
turbidity. The lowest concentration of fraction in the broth for without Abdullahi et al., 2011) suggests that the isolated turbidity was recorded as the MIC. compound (PS vlc-1) is a pentacyclic triterpene (Table 1). The 1H spectrum showed a proton quartet at δH 2.25 (H-
Minimum bactericidal concentration (MBC)/Minimum fungicidal 4) and a methyl doublet at 0.87 (H-23). Other methyls concentration (MFC) appeared as singlets at 0.71 (H-24), 0.86 (H-25), 1.06 (H- 26), 1.04 (H-27), 1.17 (H-28), 0.99 (H-29), and 0.94 (H- MBC/MFC was carried out to determine whether test microbes were 30) (Table 1). Its 13C spectrum gave a carbonyl signal at killed or only their growth was inhibited. Mueller Hinton and 213 ppm typical of a saturated ring ketone; other peaks Sabouraud Dextrose Agar were prepared according to manufacturer’s instruction, sterilized at 121°C for 15 min, and (Table 1) were also in good agreement with reported poured into sterile petri-dishes. A loop-full of the test mixture was data. The spectra data of PS vlc-1, shown in Table 1, removed from each plate, incubated at 37°C for 24 h for bacteria, 7 were used together with literature reports (Igoli and Gray, days for fungi, and was observed for the presence of colonies 2008; Abdullahi et al., 2011) to identify PS vlc-1 as which indicated growth. MBC/MFC was the lowest concentration of friedelan-3-one. Identification was further corroborated extracts that showed no bacterial or fungal growth. using its 2-D spectra (Figures 2 and 3). For instance in its proton-proton correlation spectroscopy (1H-1H COSY) (Figure 2), the compound gave a correlation between the RESULTS AND DISCUSSION protons at δH 2.25 H-4 (1 H,q) and 0.87 H-23 (3H, d) indicating a methyl substituent at position C-4 typical of a Previous investigations of leaf-extracts of P. santalinoides friedelane moiety (Abdullahi et al., 2011). In its showed that the leaf-extracts exhibited broad growth Heteronuclear Multiple Bond Correlation (HMBC) inhibition against microbes; these studies found extracts spectrum (Figure 3), the methyl at 0.87 (H-23) gave to be active against E. coli, S. typhi, S. aureus, Shigella correlation to the carbonyl at C-3. The proton at δH 2.25 flexneri, Alcaligenes faecalis, Enterobacter. aerogenes, H-4 also gave correlation to C-3 (Figure 3), a Pseudomonas aeruginosa (Osuagwu and Akomas, 2013; confirmation of a ring typical of friedelan-3-one ring A Odeh and Tor-Anyiin, 2014). The antimicrobial activities (Figure 1). results showed ethyl acetate extract to have the highest This is the first report of the presence of friedelan-3-one zone of inhibition (35 mm) at 20 mg/ml. It showed in P. santalinoides. The isolated compound (PS vlc-1) moderate activity against Gram-negative strains of was investigated for antimicrobial activity against MRSA, bacteria that are frequently reported to be less sensitive S. aureus, C. ulcerans, S. pneumoniae, C. jejuni, H. to plant extracts (Eze et al., 2012). Thus, further pylori, E. coli, S. dysenteriae, C. tropicalis and C. krusei. purification via VLC of this ethyl acetate extract yielded and had MIC values of 5 µg/ml for most pathogens (Table 11 mg of a white non-crystalline substance (PS vlc-1). 3). For MBC/MFC, it showed moderate antimicrobial Comparison of the NMR spectral features of this activity against most of the pathogens tested (Table 4). substance with existing literature (Igoli and Gray, 2008; The highest diameter of zone of inhibition was against S. 534 Afr. J. Biotechnol.
Table 1. Spectral data of PS vlc-1 in CDCl3 compared with literature reports.
Experimental data (PS vlc- Literature data Literature data
1) (Abdullahi et al., 2011) (Igoli and Gray, 2008) Position
of H/C 1H( ) 13C( ) 1H( ) 13C( ) 1H( ) 13C( ) H C H C H C 1 1.96,1.67 22.3(t) 1.95, 1.71 (2H, add) 22.3(t) 1.97 22.5 2 2.37, 2.29 41.6(t) 2.37,2.27 (2H,add) 41.5(t) 2.40, 2.28 41.8 3 - 213.3(s) - 213.2(s) - 213.0 4 2.25 58.3(d) 2.25 (1Hq) 58.2(d) 2.28 58.5 5 - 42.2(s) - 42.1(s) - 42.7 6 * 41.3(t) 1.74,1.28 (2H,d) 41.3(t) * 41.6 7 1.74 18.3(t) 1.74, 1.36 (2H,m) 18.2(t) * 18.5 8 1.38 53.1(d) 1.38 (1H,dd) 53.1(d) * 53.4 9 - 37.5(s) - 37.4(s) - 37.7 10 1.53 59.5(d) 1.53 (1H,m) 59.5(d) * 59.6 11 1.47 35.6(t) 1.45, 1.26 (2H,m) 35.6(t) * 35.9 12 * 30.5(t) 1.33, 1.32 (2H,m) 30.5(t) * 30.8 13 - 39.7(s) - 39.7(t) - 40.0 14 - 38.3(s) - 38.5(s) - 38.6 15 * 32.5(t) 1.47,1.27 (2H,m) 32.6(t) * 32.7 16 1.59 36.0(t) 1.58,1.35 (2H,m) 36.0(t) * 36.3 17 - 30.0(s) - 30.0(t) - 29.9 18 * 42.8(d) 1.56 (1H,m) 42.8(d) * 43.1 19 * 35.4(t) 1.37,1.22 (2H,m) 35.3 * 35.6 20 - 28.2(s) - 28.2(s) - 28.4 21 1.53,1.38 32.3(t) 1.50.1.31 (2H,m) 32.8(t) * 32.3 22 0.94(m) 39.3(t) 1.51.0.95 (2H,m) 39.2(t) * 39.5 23 0.87(d) 6.9(q) 0.88 (3H,d) 7.0(q) 0.89(d) J=2.7 7.04 24 0.71(s) 14.2(q) 0.73 (3H,s) 14.6(q) 0.74(s) 14.3 25 0.86(s) 18.1(q) 0.87 (3H,s) 17.9(q) 0.88(s) 18.2 26 1.06(s) 20.3(q) 1.01 (3H,s) 20.2(q) 1.01(s) 20.5 27 1.04(s) 18.7(q) 1.05 (3H,s) 18.6(q) 1.02(s) 18.9 28 1.17(s) 32.1(q) 1.18 (3H,s) 32.1(q) 1.06(s) 32.2 29 0.99(s) 35.1(q) 1.00 (3H,s) 35.0(q) 1.19(s) 32.0 30 0.94(s) 31.8(q) 0.94(3H,s) 31.8(q) 0.96(s)1.26(s) 35.2
*Overlapping signals; δChemical shift; - = No hydrogen.
pneumonia (32 mm), while the least was against E. coli and anticandidal (C. albicans, C. krusei and Candida (22 mm) (Table 2). glabrata) activities. The isolated compound (friedelan-3-one), a terpene, In a study by Ee et al. (2005), friedelin was reported to has been reported to exhibit antifeedant and anti- exhibit growth inhibitory activities against (human inflammatory activities (Duke, 1992). Friedelan-3-one has oestrogen receptor-negative breast cancer) MBA-MD-231 been found to show hepatoprotective activity (Džubák et cells. al., 2006). The mode of action of terpenes is speculated The foregoing account of medicinal properties of to involve the disruption of the cell membrane activity friedelin, indicate that it is an important medicinal (Cowan, 1999). In the present study, friedelin compound. Its isolation from the leaves of P. demonstrated antibacterial (MRSA, S. aureus, S. santalinoides demonstrates it to be a leading medicinal pneumonia, H. pylori, and E. coli) and anticandidal principle behind traditional/folkloric applications of the activity (C. tropicalis); a previous study by Kuete et al. leaves in antibacterial, antifungal, anti-inflammatory, (2007) showed friedelin to exhibit antibacterial antidiarrheal, and other infectious diseases. (Enterococcus faecalis, S. aureus and P. aeruginosa) This study demonstrates the leaves of P. santalinoides Odeh et al. 535
(a)
(b)
Figure 2. Prominent 1H-1H COSY labels (a) and correlations (b) for isolated compound.
536 Afr. J. Biotechnol.
(a)
(b)
Figure 3. Prominent HMBC labels (a) and correlations (b) for isolated compound. Odeh et al. 537
Table 2. Antimicrobial sensitivity and diameter of zone of inhibition.
Extract Vended antimicrobials Test microorganism PS-vlc-1 Sparfloxacin Ciprofloxacin Fluconazole MRSA S (26) S (37) R R S. aureus S (29) S (38) S (35) R C. ulcerans R S (35) S (37) R S. pneumoniae S (32) S (42) S (40) R C. jejuni R S (37) S (32) R H. pylori S (24) R S (34) R E. coli S (22) S (40) S (37) R S. dysenteriae R S (39) S (40) R C. tropicalis S (30) R R S (35) C. krusei S (27) R R S (37)
S: Sensitive; R: Resistance; PS-vlc-1 concentration = 40 µg/ml; Drug concentration (Positive Control) =5 µg/ml; Numeric value in brackets = Diameter of zone of inhibition in millimetres.
Table 3. Minimum inhibition concentration (MIC) of PS-vlc-1 to be a veritable source for this compound with a against test microorganism. promising antimicrobial activity.
Extract concentration (µg/ml) Test microorganism 40 20 10 5 2.5 Conflict of Interests MRSA - - ∞ + ++ S. aureus - - - ∞ + The authors have not declared any conflict of interests. C. ulcerans S. pneumoniae - - - ∞ + C. jejuni Abbreviations H. pylori - - ∞ + ++ MRSA, Methicillin resistant Staphylococcus aureus; MIC, E. coli - - ∞ + ++ minimum inhibition concentration; MBC, minimum S. dysenteriae bactericidal concentration; MFC, minimum fungicidal C. tropicalis - - - ∞ + concentration; VLC, vacuum liquid chromatography; TLC, C. krusei - - - ∞ + thin layer chromatography; PS-vlc-1, fraction of active - = No turbidity (no growth), ∞= MIC, +=turbidity (light growth) compound isolated; DMSO, dimethylsulphoxide. ++=moderate turbidity. MRSA: Methicillin Resistant Staphylococcus aureus.
REFERENCES
Abdullahi M, Kolo I, Oyewale A, Amupitan J (2011). Antimycobacterial Table 4. Minimum Bactericidal/Fungicidal Concentration friedelane-terpenoid from the root bark of Terminalia avicennioides. (MBC/MFC) of PS vlc-1 against test micro-organisms. Am. J. Chem. 1(2):52-55. Agroforestry Tree Database. (2011). A Tree Species Reference Extract concentration (µg/ml) Selection Guide. November 7, 2015, from Test microorganism File//E:speciesinformationP.santalinoides.htm. 40 20 10 5 2.5 Anowi C, Okonkwo C, Agbata C, Ezeokafor E (2012a). Preliminary MRSA - ∞ + ++ +++ Phytochemical Screening, Evaluation of Acute Toxicity and Antipyretic Activity of Methanolic Extract of Pterocarpus santalinoides S. aureus - - ∞ + ++ (Fabaceae). Int. J. Pharm. Phytopharm. Res. 1(6):343-346. C. ulcerans Anowi C, Umeokoli B, Onyegbule AF, Okonkwo C, Chibeze I (2012b). S. pneumoniae - - ∞ + ++ Analgesic, Phytochemical and Acute Toxicity Evaluation of The Methanol Extract of the Leaves of Pterocarpus santalinoides-family C. jejuni Fabacea. Int. J. Pharm. Sci. Res. 3(7):2018-2023. H. pylori - ∞ + ++ +++ Cowan MM (1999). Plant products as antimicrobial agents. Clin. E. coli ∞ + ++ +++ ++++ Microbiol. Rev. 12(4):564-582. Duke J (1992). Handbook of Biologically Active Phytochemicals and S. dysenteriae Their Activities. (B. Raton & A. Ann, Eds.). Tokyo: CRC Press. 183 C. tropicalis - - ∞ + ++ pp. C. krusei - ∞ + ++ +++ Džubák P, Hajdúch M, Vydra D, Hustová A, Kvasnica A, David M, Sarek J (2006). Pharmacological activities of natural triterpenoids and - = No colony growth, ∞=MBC/MFC, + = scanty colony growth, their terapeutic implications. Nat. Prod. Rep. 23:394-411. ++ = moderate colonies growth, +++ = heavy colony growth. Edeoga H, Okwu D, Mbaebie B (2005). Phytochemical constituents of 538 Afr. J. Biotechnol.
some Nigerian medicinal plants. Afr. J. Biotechnol. 4(7):685-688. Nworu C, Akah P, Nwachukwu J, Asogwa C (2009). Antidiarrhoeal Ee GC, Lim CK, Rahmat A, Lee HL (2005) Cytotoxic activities of activity of Pterocarpus santalinoides L’Hérit ex DC leaf extract. J. chemical constituents from Mesua daphnifolia. Trop. Biomed. complement. Integr. Med. 6(1):8-11. 22(2):99-102. Odeh IC, Tor-Anyiin TA (2014). Phytochemical and antimicrobial Eze SO, Cornelius C, Okereke HC (2012). Phytochemical and evaluation of leaf-extracts of Pterocarpus santalinoides. Eur. J. Med. antimicrobial analysis of the stembark of Pterocarpus santalinoides Plants 4(1):105-115. (Nturu ukpa). Asian J. Nat. Appl. Sci. 1(3):26-32. Osuagwu G, Akomas C (2013). Antimicrobial activity of the leaves of Hassan M, Oyewale A, Amupitan J, Abdullahi M, Okonkwo E (2004). three species of Nigerian Pterocarpus (Jacq.). Int. J. Med. Aromat. Preliminary Phytochemical and Antibacterial Investigation of Crude Plants 3(2):178-183. Extracts of The Root Bark of Detarium microcarpum. J. Chem. Soc. Phillipson JD (2001). Phytochemistry and medicinal plants. Niger. 29(1):26-29. Phytochemistry 56(3):237-243. Igoli J, Gray A (2008). Friedelanone and other triterpenoids from Sofowora A (1982). Medicinal plants and traditional medicine in Africa. Hymenocardia acida. Int. J. Phys. Sci. 3(6):156-158. pp. xviii + 256 pp. Igoli J, Igwue I, Igoli N (2003). Traditional Medicinal Practices Among Tor-Anyiin TA, Igoli J, Anyam JV, Anyam JN (2015). Isolation and The Igede People of Nigeria. J. Herbs Spices Med Plants 10(4):1-10. antimicrobial activity of sarracenin from root bark of Strychnos Keay R (1989). Trees of Nigeria. Clarendon Press: Oxford, UK. New spinosa. J. Chem. Soc. Niger. 40(1):71-75. York. Kuete V, Komguem J, Penlap BV, Meli AL, Tangmouo JG, Etoa FX, Lontsi D (2007). Antimicrobial components of the methanolic extract from the stem bark of Garcinia smeathmannii Oliver (Clusiaceae). S. Afr. J. Bot. 73(3):347-354.
Vol. 15(14), pp. 539-548, 6 April, 2016 DOI: 10.5897/AJB2015.14980 Article Number: 95C8D3F57894 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Nanoformulation and antibiotic releasing property of cefotaxime nanoparticles
Maysaa Chasib Al-Moahmmedawi
Scholar Rescue Fund- Institute of International Education (IIE) USA.
Received 17 September, 2015; Accepted 10 March, 2016
The objective of this study was to design nano-antibiotic to enhance their release from biomaterial agents. Cefotaxime was used as a model antibiotic substance in this carrier system. These nanoparticles were preformulated using different concentrations of polycaprolactone (PCL) and poly (vinyl alcohol) as coating material and prepared using double emulsion solvent evaporation method. The physiochemical properties of cefotaxime nano-antibiotic (Cefo-NPs) stability were determined. Results showed that the encapsulation efficiency of nanoparticles increased with increase in polymer concentration. In addition, dynamic light scattering (DLS) and atomic force microscope (AFM) indicated that the particles size were in the range of 189 to 219 nm. The drug release profile of Cefo-NPs shows rapidly the release behaviour under acidic environment. And thus make it a promising tool for control bacterial infection.
Key words: Polycaprolactone, poly (vinyl alcohol), cefotaxime, nanoparticle.
INTRODUCTION
Cefotaxime is a water soluble semisynthetic third unique features that influence certain types of bacteria generation of cephalosporin antibiotics, used for serious (Sivaraman and Ramamurthi, 2013). Additionally, infections caused by susceptible strains of micro- different approaches were used to combine antibacterial organisms in lower respiratory infections, genitourinary materials to achieve desirable effects. infections, gynaecologic infections, skin infections, and Nanocarriers, such as pH-sensitive nanoparticles, may central nervous system infections (Morelli, 2003). It acts represent one of these approaches that constitute an by inhibiting bacterial cell wall synthesis. alternative strategy to overcome the difficulties that are Recent studies focused on using composite related to microbial resistance and poor oral nanomaterials of silver-chitosan, chitosan-arginine, zinc- bioavailability (Ankola et al., 2010). It was found out that iron oxide, and polymer-antibiotics to control bacterial sustained release formulations prolong the action of the colonization and infection and potentially could be used drug for a long period of time and also decrease the as future therapeutic agents (Huang et al., 2011a, 2011b; frequency of drug dosage. A sustained release Lellouche et al., 2012). Although, their mechanism of formulation reduces the frequent drug administration action is still unclear, but each of these materials has (Shekhar et al., 2010).
*Corresponding author. E-mail: [email protected], [email protected]. Tel: +61 478795442.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 540 Afr. J. Biotechnol.
Table 1. Formulation plan or cefotaxime nanoparticles.
Nanoformulation Cefotaxime (mg/ml) PCL polymer (w/v %) PVA (w/v %) Cef-NP-1 5 1 0.2 Cef-NP-2 5 2 0.2 Cef-NP-3 5 1 0.3 Cef-NP-4 5 2 0.3
Poly ε-caprolactone (PCL) is a synthetic, biodegradable, Percentage yield biocompatible polymer often used in the formulation of nanoparticles. Nanoparticles prepared using PCL as a The percentage practical yield was calculated to know about the percent yield or efficiency of any method, which would help in the matrix was found to be larger than that prepared with selection of appropriate method of production. Practical yield was other polymers. The colloidal suspension exhibited calculated as the weight of the dry nanoparticles recovered from sustained release profile (Kumari et al., 2010). Since it is each batch in relation to the sum of the starting material (Ramteke a low cost material, it is approved by the US Food and et al., 2006). Drug Administration, and experience’s slow degradation in the body (Mora-Huertas et al., 2010; Haddadi et al., Percentage yield (PY) = (Practical yield/Theoretical yield) × 100
2008). The aim of this study was to prepare and characterize new nanoformulation of cefotaxime with PCL Fourier transform infra-red spectroscopy study using solvent evaporation method. The Fourier transform infrared (Shimadzu-FTIR, Mo-del-8000 provided by Chemical Department, College of Science, Al-Nahrain MATERIALS AND METHODS University, Baghdad, Iraq) analysis was conducted to verify the possibility of chemical bonds between drug and polymer. Samples of pure cefotaxime, pure PCL and Cefotaxime-PCL physical mixture Preparation of standard curve of cefotaxime -1 1:1 were scanned in the IR range from 400 to 4000 cm with From the stock standard solution, aliquots of 2, 4, 6, 8, and 10 ml carbon black as reference. The detector was purged carefully by were pipette out and the volume was made upto 10 ml with clean dry helium gas to increase the signal level and reduce phosphate buffered saline (PBS) pH 7.4 to obtain concentrations in moisture. the range of 20 to 100 μg/ml. The absorbance of these solutions was measured at 256 nm by UV-Visible spectrophotometer, using phosphate buffer of pH 7.4 as blank. The absorbance values were Encapsulation efficiency (EE %) plotted against concentration to obtain the standard graph. The encapsulation efficiency, (EE %), was measured at wavelength of 256 nm by UV spectrometer (Spectronic Genesys 10 Bio, Formulation of cefotaxime nanoparticles (Cefo-NPs) Thermo Electron Cooperation, WI, USA). The standard curve was prepared using drug concentration ranging from 1 to 10 mcg/ml and 2 Cefotaxime loaded nanoparticles were prepared with water in oil in had a regression equation of y = 0.076x with R = 0.988. In each water (w/o/w) double emulsion- solvent evaporation method. In this sample, EE% was measured by separating the aqueous phase with method, cefotaxime equivalent to 5% w/v was dissolved in 500 μl of the colloidal one after centrifuge at 14,000 rpm for 30 min (VWR PBS (0.01 M, pH 7.4) to form cefotaxime aqueous solution. The micro 18R, VWR Inc., West Chester, USA). The encapsulation cefotaxime aqueous solution was emulsified in an organic phase efficiency of the drug loading was calculated using the following consisting of 1 to 2% of the PCL polymer inorganic solvent di-cholo equation (Shabouri, 2003): methane (DCM) to form primary water in oil emulsion. The emulsion T F T was further emulsified in an aqueous 12.5 ml of poly-vinyl alcohol Encapsulation efficiency (%) = (A –A ) /A ×100 (PVA) stock solution (100 ml 2 to 3% w/v), as shown in Table 1, to T F form w/o/w emulsion. The emulsification was carried out using a where A is the total drug amount and A is the nano encapsulated micro tip probe sonicator (VC 505, Vibracell Sonic, Newton, MA, drug amount. USA) set as 55 W of energy output for 5 min over an ice bath by adding the primary emulsion in dropwise to the 20 ml of phosphate buffer (0.01 M, pH 7.4). The emulsion was stirred for 18 h on a Particle size analysis magnetic stir plate at room temperature to allow the evaporation of the organic solvent. Further 1 h vacuum drying was also performed The size distributions along the volume mean diameters of the to remove any residual organic solvent present. Any excess amount suspending particles were measured by dynamic scattering particle of PVA was removed by ultracentrifugation at 16000 rpm at 40°C size analyser (Nanotrac Particle Analyzer 150, Microtrac Inc., PA, for 20 min (Remi, India) followed by washing with double distilled USA) (Alexis et al., 2008). water. The supernatant was collected and kept for an estimation of the amount of the drug which was not encapsulated. The recovered nanoparticulate suspension was lyophilized for two days (-800°C Determination of drug release profile of Cefo-NPs and <10 mm mercury pressure, LYPHILOCK 12, Labconco, Kansas City, MO, USA) to provide the lyophilized powder for further use One millilitre of the aformentioned Cef-PCL nanoparticles was (Niwa et al., 1994). centrifuged for 45 min at 20,000 rpm. After that, the precipitated Al-Moahmmedawi 541
Table 2. Practical yield of nanoparticles.
Formulation Practical yield (mg) Total amount of ingredients (mg) Percentage yield (%) Cef-NP1 54 84 64.2 Cef-NP2 48 64 75 Cef-NP3 45 57 78.9 Cef-NP4 54 98 56.1
Figure 1. Standard calibration curve of Cefotaxime in phosphate buffer saline (7.4) at 256 nm.
Cef-PCL was redispersed in 1 ml of buffer solutions at pH 1.5, 4.5, water-soluble cefotaxime drug (Kim et al., 2002). Four 6.8, and 7.2. The dispersed particles were incubated for 1, 2, 4, 6, formulations of Cefo-NPs were formulated using different 12, 24, 48, 72 and 96 h at 37°C, which was conducted in triplicate. polymer emulsifier ratios, as shown in Table 2. The The amount of released drug was measured at 256 nm by UV- spectrometry. These results were shown as average ± standard formulations are subjected to evaluation parameters like deviation (n= 3). In addition, the drug loading efficiency (7.2 wt.%) particle size, surface topography, drug encapsulation, was measured in the same manner. Briefly, cefotaxime weight was and zeta potential. measured after lyophilisation and then dissolved in 1 ml of distilled Physicochemical characteristics, such as particle size water. The loaded amount of drug was measured by UV- and surface properties, all represented important spectrometry, using the following formula (Alexis et al., 2008): parameters for determining the in vitro drug release, Drug loading efficiency = (Weight of drug in nanoparticles/Weight of cellular uptake, and cytotoxicity of nanoparticles. The in PCL-PVA) × 100. vivo pharmacokinetics and biodistribution, influenced the therapeutic efficacy of the encapsulated drug (Youan et al., 2001). The absorbance measurement of cefotaxime RESULTS AND DISCUSSION standard solution containing 20 to 100 mcg/ml of drug in pH 7.4 PBS as shown in Figure 1 presented the standard The w/o/w multiple emulsion solvent evaporation method calibration curve. The curve was found to be linear in the is the mostly used technique for encapsulation of water- range of 20 to 100 mcg/ml at λmax 256 nm. The soluble drug. And it was the method of choice for the regression coefficient value was found to be 0.9887. 542 Afr. J. Biotechnol.
Table 3. Encapsulation efficiency of cefotaxime.
Absorbance of drug Absorbance of Encapsulation Sample No. Mean (±) *STDEV **STDREV loaded (O.D.) free drug (O.D.) efficiency (%) Cef-NP1 3.097 3 74.57 3.0485 0.068589 0.0485 Cef-NP2 3.019 2.987 90.77 3.003 0.022627 0.016 Cef-NP3 3.126 2.969 58.84 3.0475 0.111016 0.0785 Cef-NP4 3.098 2.965 61.65 3.0315 0.094045 0.0665
*STDEV: Standard deviation; **STDER: standard error.
Figure 2. Drug encapsulation efficiency of nanoparticles.
Optimization of polymer and emulsifier ratio formulations. It was observed that the encapsulation efficiency increases with the increase in concentration of For enhanced drug encapsulation, the results of this polymer in the formulations. The maximum encapsulation study showed that the drug/polymer and surfactant ratios was 90.77 and 74.57% found in Cef-NP2 and Cef-NP1, and the concentration values influenced the final respectively. Furthermore, the encapsulation efficiency properties of the prepared nanoparticles. For PVA- increases as PVA concentration also increases on emulsified PCL nanoparticle formation, drug concentration increasing the concentration of internal phase as was kept constant at 5 mg per formulation, and PCL indicated in Table 3 and drug content in Figure 2. concentration varied from 1 to 2 g to give a drug/polymer In this study, the result shows that encapsulation ratio of 1:10 and 1:20. And for PVA concentration, it was efficiency of the drug was depended on the solubility of from 1:1 and 1:2. These two different ratios of PCL and the drug in the solvent and continuous phase. A similar poly vinyl alcohol were applied. In this way, four observation was reported by Pignatello et al. (1997). The formulations were prepared. The percentage of nano- reason could be due to increase in size to encapsulate particles yields varied from 55 to 54 mg corresponding to more drug and more surfactant similarly speed up the the amount of total amount of ingredients from 57 to 98 encapsulation process by enhancing the binding contact mg shown in Table 2. The results showed that the between drug and polymer in emulsion stage. percentage of practical yield increased as the amount of They are two important factors effect on the polymer added to each formulation increased, although it encapsulation efficiency, the type of polymer with the may not be dependent upon PVA concentration in the concentration and organic solvent selection. Large size formulation. Maximum yield was found to be 75 % for nanoparticles are produced whenever high concentration Cef-NP2 and 78.9% for Cef-NP3. of polymer in organic phase is applied (Dey et al., 2009).
An interesting study found out that the stability of the Drug encapsulation efficiency emulsion droplets has an effect on the size of the formulated nanoparticles which is affected by miscibility The drug content in four batches of Cefo-NPs was of organic phase with water (Lourenço and Pinto, 2009). studied. Table 3 and Figure 2 show the results of the In this study, using dichloromethane as a water immiscible drug encapsulation efficiency in each of these solvent led to the formation of nanoparticles by a true Al-Moahmmedawi 543
Table 4. Size distribution of nanoparticles formulation shown in Figures 3 and 4. The lactam (C=O) band with zeta potential. appears at 1780 cm-1 in the spectrum of cefotaxime, while the overlapped amide and ester (C=O) bands Nanoformulation Particle size (nm) Zeta potential (mV) appear at 1640 cm-1; the complexes show these bands at Cefo-NP1 204 -45 (±) 0.78 around 1720 to 1740 and 1630 to 1650 cm-1 ranges, Cefo-NP2 219 -58 (±) 1.2 respectively. All this suggests that coordination of the Cefo-NP3 209 -34 (±) 3.1 ligand occurs through the oxygen atom from the lactam Cefo-NP4 189.8 - 38 (±) 0.96 carbonyl group rather than the amide and ester carbonyl groups. The lactam carbonyl bands were substantially shifted toward lower frequencies (40 to 60 cm-1) relative
to the value of the uncomplexed cefotaxime, while in the emulsification mechanism in which the larger emulsion overlapped amide and ester carbonyl bands, the shifting droplets are broken into smaller droplets by the was not significant (Chernysheva et al., 2003). application of external energy.
Atomic force microscopy (AFM) of Cefo-NPs Particle size and Zeta potential The AFM images showed that the nanoparticles formed The particle size of all formulated nanoparticles was as a result of solvent evaporation were spherical shaped estimated and was found to be in the range of 180 to 220 and smooth in surface morphology, with an average nm which indicates that they are stable. As the diameter of less than 200 nm (Figure 5).The topography concentration of polymer increased, the particle size also of cefotaxime nanoparticles were observed using AFM increased. Furthermore, when the concentration of and it was found that the average diameter for Cef-NP1 stabilizer (PVA) was increased, there was a decrease in was 109.8 nm, while it was 90.7 nm for Cef-NP2 the particle size from 219 to 189 nm. The polymer formulation. In addition, the AFM of Cef-NP3 and Cef- concentration in the internal phase is a crucial factor in NP4 nanoparticles were 103.9 and 74 nm (cumulative increasing the size of nanoparticles. It was found out that data details not published), respectively. Results showed high viscosity of the dispersion leads to higher resistance no significant adsorption at the organic/aqueous phase shear forces of emulsification. Therefore, at high viscous boundary when DCM was applied as organic solvent. dispersions, the globule size of emulsions obtained will And thus, resulting in higher interfacial tension, causing be higher, resultantly; after evaporation of solvent, higher agglomeration to occur, and hence the bigger particle size of nanoparticles were obtained. Earlier studies found sizes (Chernysheva et al., 2003). All the nanoparticles out that particle size was proportional to the viscosity of prepared with DCM as solvents were not uniform. the dispersed phase (Kim et al., 2002; Si and Samulski, Sample preparation plays an important factor in order 2008). to get useful AFM images (Song et al., 2006). Samples The stability study of the nanoformulation was must be thin enough and must adhere well to the surface, performed by measuring the zeta potential of the otherwise, the scanning process will producing artefacts. nanoparticles using the zeta meter ± 3M. The results are More details are presented in Chicea et al. (2012). In evaluated in Table 4. order to prepare the sample, a drop of nanofluid was The significance value of using zeta potential is that its deposited on a freshly cleaved mica substrate and value can be related to the stability of colloidal dispersion. stretched with a blade to form a very thin layer. The thin The zeta potential indicates the degree of repulsion bet- layer was left for 3 h to evaporate. The sample was ween adjacent, similarly charged particles in dispersion. attached to the AFM plate. First, a large area (5 μm × 5 For molecules and particles that are small enough, a high μm) surface scan was carried out. Since the resolution zeta potential will confer stability, that is, the solution or used in the first scan is not good enough to image dispersion will resist aggregation (Anacona and Silva, nanoparticles on a surface, several scans were done to 2005). select a flat area on the surface where there appear to be singular nanoparticles rather than aggregates, which were present, as well. Finally, a bigger resolution scan Fourier transform infrared spectroscopy (FTIR) (512×512 pixels) was achieved and the topography is as
shown in Figure 5. The scanned area is 1.0 μm × 1.0, Preformulation studies were carried out to study the several aggregates nanoparticles located on the scanned compatibility of cefotaxime with PCL prior to the area is noticed. preparation of cefotaxime loaded nanoparticles. Infrared spectra of pure cefotaxime and PCL and their combination were studied. The FTIR studies show that there was no In vitro drug release of Cefo-NPs interaction between the drug and the polymer used. As indicated in the Figure 4. FTIR spectra of cefotaxime and In vitro release profile of formulations is as shown in its complex are similar and the main frequencies are Figure 6. All the selected formulations showed sustained 544 Afr. J. Biotechnol.
Figure 3. Zeta potential distribution curve of four formulated nanoparticles: (A) Cefo-NP1, (B) Cefo-NP2, (C) Cefo-NP3, and (D) Cefo-NP4. Al-Moahmmedawi 545
Figure 4. FTIR spectra of cefotaxime nanoparticle (NP-Cefo), Cefotaxime, Polycaprolactone-poly vinyl alcohol (PCL-PVA), and Polycaprolactone (PCL).
Figure 5. AFM plates for nanoformulation of cefotaxime: (A) Cefo-NP1, (B) Cefo-NP2, (C) Cefo-NP3, and (D) Cefo- NP4. 546 Afr. J. Biotechnol.
Cumulative release % Cumulative release %
Time (h) Time (h)
Cumulative release % Cumulative release %
Time (h) Time (h)
Figure 6. Cumulative % drug release vs. time for formulation of cefotaxime nanoparticles: A. pH (1.5), B. pH (4.5), C. pH (6.8), D. pH (7.4).
release patterns of drug in just 2 h. Except Cef- release in 12 h. From the results of data fitting to (pH 1.5) with about 60% of drug release within 6 NP1 showed controlled release up to 8 h. Cef- various models, Cef-NP4 formulation shows h. Whereas only 30% was released at lower pH NP4 formulation showed 95.37% release after 6 controlled release. Cefotaxime was abruptly conditions (pH 1.5 and 4.5) during the same time h. Whereas CCef-NP2 batch showed 94.29% released from Cef-NPs under acidic conditions period and both release profiles showed sustained Al-Moahmmedawi 547
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Vol. 15(14), pp. 549-556, 6 April, 2016 DOI: 10.5897/AJB2015.14928 Article Number: C814CEF57896 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Polymorphism of growth hormone gene and its association with wool traits in Egyptian sheep breeds
Ibrahim M. Farag1, Ahmed M. Darwish1, Hassan R. Darwish1, AbdelAziz K. B.1, Ramadan W. A.2, Mohamed M.I.3 and Othman E. Othman1*
1Cell Biology Department, National Research Centre, Dokki, Egypt. 2Wool Production and Technology Department, Animal and Poultry Division, Desert Research Centre, Egypt. 3Animal Production Department, National Research Centre, Dokki, Egypt.
Received 19 August, 2015; Accepted 11 March, 2016
Growth hormone (GH) gene has been described as a candidate gene for marker-assisted selection in different farm animals. The present study was designed to identify the polymorphism in GH gene and its association with variation of wool traits in Egyptian sheep breeds. Wool and blood samples were collected from 42 animals including two breeds (Barki and Rahmani) and one crossbred (Rahmani x Awase). Measurements of wool traits were analyzed and involved staple strength (Str), staple length (STL), fiber diameter (FD) and clean fleece yield (CFW). DNA was extracted from blood samples and a 365-bp fragment from exon V was amplified by polymerase chain reaction (PCR). Single strand conformation polymorphisim (SSCP) analysis showed two conformational patterns. The pattern I was recorded to be more frequent (83.3, 92.86 and 90%) than pattern II (16.7, 7.14 and 10%) in Barki, Rahmani and crossbred, respectively. The sequence analysis showed one single nucleotide polymorphism (C/T). The pattern I (allele T) has been found to affect CFW and FD than pattern II (allele C). Whereas, C allele was more pronounced for Str and STL. These traits are the most important parameters determining commercial values of wool that are preferred for clothing or carpets industry. The nucleotide sequences of C and T alleles were submitted to GenBank and have the accession numbers: KT250511 and KT250512, respectively. In conclusion, the present results provide evidence that there is a single nucleotide polymorphism within GH gene in Egyptian sheep breeds. This mutation was found to have some effects on wool traits. Therefore our data show interesting prospects in future selection programs for improving wool industry.
Key words: Sheep, wool, growth hormone (GH) gene, polymorphism, single strand conformation polymorphism (SSCP).
INTRODUCTION
Wool is widely used throughout the world by clothing and dependent on the quality of wool fibers, but considerable carpets industry. The efficiency of wool processing is diversity exists both within and between fleeces and this
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 550 Afr. J. Biotechnol.
is observed even in inbred lives of sheep (Itenge-Mweza as PCR- single strand conformation polymorphisim et al., 2007; Itenge et al., 2010). This variation might be (SSCP) technique (Santos et al., 2004). However, several better controlled by using the genetic markers that studies proved that SSCP analysis is an effective contribute to the wool fiber (Itenge et al., 2010). technique for the detection of polymorphisms, (Sheffield The use of genetic markers in the wool industry would et al., 1993; Neibergs et al., 1993; Barroso et al., 1999). allow earlier assessment of an animal's potential and Comparing with other methods, for instance, denaturing increase the accuracy and efficiency of selection (Itenge- gradient gel electrophoresis, temperature gradient gel Mweza et al., 2007). Several studies suggested that the electrophoresis and chemical and ribonuclease cleavage, growth hormone (GH) gene may influence wool quality SSCP has several advantages; it does not require and quantity (Sami et al., 1999; Beh et al., 2001; Yousefi specific equipment, it is technically simpler and faster, it and Azari, 2012). GH gene plays an important role in can be used in most laboratories and is not very growth regulation and development in sheep (Boyd and expensive. Thus, SSCP analysis is the technique of Bauman, 1989; Bathaei and Leory, 1998). The ovine GH choice when screening for point mutations and minor is about 1.8 kb long and contains five exons and four deletions within a given fragment is concerned (Neibergs introns (Hajihosseinlo et al., 2013). Ayuk and Sheppard et al., 1993). Therefore, the present study was designed (2006) reported that GH is an anabolic hormone to identify potential variation in GH gene using PCR- synthesized and secreted by the somatotroph cells of the SSCP method and its association with wool traits in anterior lobe of the pituitary gland in a circadian and Egyptian sheep breeds. pulsatile manner. Boyd and Bauman (1989) found that the effects of GH on growth were in several tissues including bone, muscle and adipose tissue. These MATERIALS AND METHODS authors observed that these effects result from both Sheep direct action of GH on the partition of nutrients and cellular multiplication as well as IGF-1 mediated action This study used two breeds and one cross-bred of Egyptian stimulating cell proliferation and metabolic processes and domestic sheep. These two breeds are Barki (7 males and 11 associated with protein deposition. females) and Rahmani (7 males and 7 females), whereas, the crossbred was Baladi x Awase (10 females). Barki breed were Generally, in farm animals, the detection of the patterns maintained at Nubaria Farm, National Research Centre, Egypt. (polymorphisms) of GH gene might play an important role Rahmani sheep were sourced from two Animal Production Farms. in growth (Breier, 1999), development (Akers, 2006), These farms belong to Faculty of Agriculture, Ain Shams University lactation (Baldi, 1999), reproduction (Scaramuzzi et al., and Faculty of Agriculture, Al-Azhar University, Egypt. Crossbred 1999) and metabolism (Bauman, 1999). Pereira et al. (Baladi x Awase) samples were collected from South Sinai (Special (2005) reported that the polymorphism of GH gene in the Farm, Egypt).
Canchim beef cattle, significantly had effects on yearling weight (YW) (P < 0.05); these effects were positive and Blood sample collection and DNA extraction associated with Leucine/Valine (L/V). Also, several studies in bovine, found that there are correlation Blood samples were collected from all selected animals by vacuum between polymorphism in GH gene and milk traits (Peel glass tubes containing EDTA-Na2 as an anticoagulant reagent. The and Bauman, 1987; Lucy et al., 1993; Hoj et al., 1993; whole blood samples were stored at -20°C until the time of DNA extraction. DNA was extracted from 100 µl of blood as described by Ng-Kwai-Hang, 1997). These researchers showed that Boom et al. (1990). After estimating the DNA concentration and its the variations of GH gene are considered to be molecular purity by spectrophotometer, DNA was diluted in sterile water to be markers for molecular assisted selection to increase milk a final concentration of 50 ng/ul before PCR amplification. DNA was production and improve milk composition. In sheep, Allain also examined by loading samples on 0.7% agarose gel and et al. (1998) reported that GH gene is located on visualizing the band under gel documentation system. chromosome 3, and found segregation for coefficient of variation of wool traits (fiber diameter and staple length) PCR amplification of the GH locus on chromosomes 3 and 4 in a composite sheep line (INRA401). Also, Yousefi and Azari (2012) reported three The 365-bp fragment of the ovine GH gene was amplified. Based different conformational patterns in exon V of the GH on the ovine GH gene sequence, one pair of oligonucleotide gene in Zel sheep breed at frequencies of 19% for primers were designed to amplify this fragment using the primer 5.0 software (www.primerbiosoft.com). These sequences of the primers pattern 1 (G1), 51% for pattern 2 (G2) and 30% for used in PCR were F: 5'-GAAACCTCCTTCCTCGCCC-3' and R: 5'- pattern 3 (G3). These results showed that there was a CCAGGGTCTAGGAAGCCACA-3'. These sequences had also significant (P < 0.05) effect of pattern 1 (G1) on staple previously been reported by Barracosa (1996). To obtain 365 bp strength of wool traits. fragment of GH exon V, the following PCR mix was used. Each The polymorphisms of ovine GH have been reported by reaction mixture consisted of 12.5 µl of the master mix, 1 µl of the utilizing different techniques including restriction fragment DNA solution (50 ng/µl), 1 µl of each primer (5 pmol) and some de- ionized water making up a final volume of 25 µl. The protocol of length polymorphism (RFLP) using restriction endo- PCR amplification was used according to Yousefi and Azari (2012) nucleases TaqI and PvuII (Gootwine et al., 1996; Ofir and as follows: An initial denaturation step at 95°C for 5 min followed by Yossefi, 1996) and EcoRI (Gootwine et al., 1998) as well 35 cycles of denaturation at 95°C for 1 min, annealing at 62°C for Farag et al. 551
50s and extension at 72°C for 90s and a final extension of 72°C for 1 2 3 4 5 6 7 10 min.
Single-strand conformation polymorphism analysis
This analysis was done according to Yousefi and Azari (2012) as follows: 5 µl of each amplified product was added to 15 µl of denaturizing solution (95% formamide, 10 mM NaOH, 0.05% xylene cyanol and 0.05 bromophenol blue, 20 mM EDTA). The samples were heat-denatured at 95°C for 5 min, chilled on ice and loaded onto 8% polyacrylamide gel (39:1). The gels were run at 249-280 V for 6-8 h at 4°C. The electrophoresis was carried out in a vertical unit in 1 x TBE buffer. Gels were silver-stained according to the method of Sanguinetti et al. (1994).
Sequence analysis
PCR products representative of different SSCP patterns at GH gene exon 5 were purified and sequenced by Sigma Corporation. Sequence analysis and alignment were carried out using ClustalW2. The nucleotide sequences of detected patterns of GH gene in Egyptian sheep were submitted to GenBank (NCBI, BanKit).
Measurements of wool traits Figure 1. Representatives of PCR-SSCP analysis of 365 Wool samples collection bp amplimer of GH gene showing two patterns identified in Barki males of sheep animals. Lanes 1, 2, 4, 5, 6 and 7: Pattern I. Lane 3: Pattern II. Wool samples were obtained from the left mid-side position of each animal. Ten staples were taken from each greasy sample and used for measuring the percentages of staple length, fiber diameter, staple strength, clean wool yield (El-Gabbas, 1998) and amino acid analysis. These measurements are as follows: Statistical analysis
Staple length: Staple length was the average of 10 staples; Analysis of variance was carried out for the data to split the total measurements were made from the base to the dense part of the variance into its components by the least square technique using tip of the staple to the nearest 0.5 cm (Chapman, 1960). the general linear model procedure of SAS (2004). Comparisons among subclass means were also carried out following Tukey test (SAS, 2004). Fiber diameter: Short sections of at least 300 fibers were prepared and mounted in paraffin oil on glass slides and covered with glass cover using the method adopted by ASTM (1974). Fiber diameter was estimated using a microscope and image captured by image RESULTS analysis software (Video Pro, Leading Edge Ltd, S. Aust.) and device (LEICAQ 500 MC) with lens 4/0.12. The average fiber The PCR-SSCP analysis of 365 bp amplified fragments diameter (FD) and standard error (SE) of each sample were of GH gene revealed two patterns in forty-two tested calculated. Egyptian sheep animals (Figure 1). Pattern I was recorded with high frequency in all investigated sheep in Clean wool yield (YLD %): Determination of clean wool weight for this study as shown in Table 1. Its frequency in Barki each sample was carried out by using the method suggested by Chapman (1960) as follows: males and females were 85.7 and 81.8%, respectively, whereas its frequency in Rahmani sheep was 85.7% in males and 100% in females. Cross-bred females (Baladi Weight of clean scoured and dry wool Clean scoured yield = × 100 X Awase) recorded 90% of pattern I. Rahmani females Weight of greasy wool possessed the highest frequency of pattern I (100%). On the other hand, the results showed the presence of Staple strength: Staple strength was estimated by measuring the pattern II with few frequencies in all tested sheep. Its force required to break the staple in Newton and dividing this value frequency in Barki males and females were 14.3 and by the thickness of the staple in Kilotex. Two staples have been plucked at random from each greasy sample to be prepared for 18.2%, respectively. Also, the frequency of pattern II in measuring their strength using the Agritest Staple Breaker (Caffin, Rahmani sheep was 14.3% in males and 0.0% in 1980) and to be in harmonious with the procedure displayed by El- females. The females of crossbred (Baladi x Awase) Gabbas (1999). possessed 10% of this pattern II. 552 Afr. J. Biotechnol.
Table 1. The pattern frequencies of GH gene in two breeds and one crossbred of tested sheep animals.
Pattern frequencies Breeds No. of animals Pattern I Pattern II No Frequency No Frequency Barki males 7 6 85.7% 1 14.3% Barki femals 11 9 81.8% 2 18.2% Total Barki 18 15 83.3% 3 16.7% Rahmani males 7 6 85.7% 1 14.3% Rahmani females 7 7 100% 0 0.0% Total Rahmani 14 13 92.86% 1 7.14% Crossbred females (Baladi x Awase) 10 9 90% 1 10%
Pattern (I) =T
Pattern (II) =C
Figure 2. The sequences of the two different patterns.
The sequence analysis showed that there is one single Table 2. Although there was only significant differences nucleotide polymorphism (SNP) (C/T) in the amplified between pattern I (allele T) and pattern II (allele C) for fragment (365-bp) of GH gene exon 5 (Figure 2). The staple length, however, the present results showed that sequence alignment between the two detected patterns the CFW and FD traits have been found to be more showed the presence of this nucleotide substitution at associated with pattern I (allele T) than pattern II (allele position 22 of the amplified fragments (Figure 3). The C). On the other hand, the STL and STR traits were more nucleotide sequences of C and T alleles were submitted pronounced in pattern II (allele C) as compared to pattern to GenBank and have the accession numbers: KT250511 I (allele T). and KT250512, respectively.
DISCUSSION The association between wool traits and GH polymorphism PCR-SSCP is used as a one of the preferred methods for screening samples to detect polymorphism on genetic Statistical analysis of wool traits and their association marker (Itenge-Mweza et al., 2007; Itenge et al., 2009). In with different detected alleles of GH gene is given in this technique, regions of the gene of interest are Farag et al. 553
Figure 3. Sequence alignment between two detected patterns.
Table 2. Least square mean of wool characteristics for different sheep breeds according to their gene pattern.
Genotype STL FD CFW STr PI (37 animals) 4.12B+0.26 29.92A+0.78 70.79A+1.39 27.89A+2.17 PII (5 animals) 5.80A+0.69 29.63A+2.11 68.20A+3.79 39.08A+5.82
*Means with different capital superscript are differ significantly within the same column. PI = pattern I (allele T), PII = pattern II (allele C), STL: staple length, FD: fibers diameter, CFW: clean fleece weight, STr: staple strength.
amplified using PCR and the products denatured and crossbred of Egyptian domestic sheep. then cooled rapidly to promote the formation of The results showed two patterns of GH gene in tested secondary structures due to internal base-pairing, which animals with higher frequency of pattern I as compared to are in turn sequence dependent (Orita et al., 1989; Vignal pattern II. Current results of GH polymorphism are similar et al., 2002). The folded single-strand DNA molecules are to that reported in previous study by Bastos et al. (2001) separated by polyacrylamide gel electrophoresis under on Portuguese indigenous sheep breed "Churra da Terra non-denaturing conditions. Molecules that differ by even Quente". These authors identified two conformational a single nucleotide may form different conformers under patterns using SSCP analysis of exon 4 of the GH gene a given set of conditions and upon electrophoresis in a and they also observed five different conformational non-denaturing polyacrylamide gel, migrate differently patterns in exon 5 of the same gene. Also, five ovine GH (Orita et al., 1989; Vignal et al., 2002). So, those exons were analyzed by Marques et al. (2001) using differences can be observed as a shift in the PCR-SSCP in 200 Portuguese Serrada Estrela ewes. electrophoretic mobility (Hayashi, 1991). Also, Hayashi Their results revealed that all exons with the exception of (1991) estimated PCR-SSCP analysis sensitivity exon 1 were polymorphic. Shiri et al. (2006) found three (probability of detecting at least one strand shifted) as conformational patterns in exon 4 of GH gene in Kordian more than 99% for 100 to 300 bp fragments and 89% for sheep. Tahmorespoor et al. (2011) and AhaniAzari et al. 300 to 450 bp fragments. Thus, PCR-SSCP technique (2011) revealed three conformational patterns using the was used in this study to detect the polymorphism in SSCP method in exon 5 of GH gene in Balouchi and exon 5 of GH sheep gene in two breeds and one Dalagh sheep breeds. Yousefi and Azari (2012) reported 554 Afr. J. Biotechnol.
three different conformational patterns (alleles) in exon 5 be the first report on detection of the single nucleotide of the GH gene located on chromosome 3 in Zel sheep polymorphism C/T at GH gene exon 5 of the sheep. In breed, 19% for pattern I (G1), 51% for pattern 2 (G2) and another study, a nucleotide change C/T was detected in 30% for pattern 3 (G3). Baluchi sheep breed at nt 271 of IGF – 1 gene. The same In the present study, the association between mutation (in IGF – 1 gene) has been detected in 8 polymorphism in GH gene and variation in wool traits different sheep breeds selected throughout Europe were also observed. The CFW and FD traits have been (Pariset et al., 2006) and in three Italian dairy sheep shown to be more associated with pattern I than pattern (Scata et al., 2010). These researchers found that allele II. On the other hand, the STL and STr traits were T exerted a positive effect on maintaining a constant milk pronounced in pattern II as compared to pattern I. The yield level during lactation. In the present study, allele T effect of GH polymorphism on wool traits was reported in in GH gene exon 5 has been found to be affected by some other studies. For example, Yousefi and Azari CFW and FD traits than C allele. Whereas, C allele has (2012) observed a significant effect (P <0.05) between been shown to be major agent for increasing the STr and polymorphism at GH gene and wool staple strength in Zel STL traits. These traits are the most important sheep. Also, in previous study, Allain et al. (1998) found a parameters determining the commercial values of wool segregation for coefficient of variation of wool traits (fiber that are preferred for clothing or carpets industry (Itenge- diameter and staple length) on chromosomes 3 and 4 in Mweza, 2007). Also, the association between INRA40/sheep, where GH gene is located on polymorphism in GH gene area (that are located on chromosome 3. chromosome 3) and variation in fibre diameter and staple However, several studies in sheep and bovine showed length has been reported in a composite sheep line the effect of GH polymorphism on other animal production (INRA 401) (Allain et al., 1998). Moreover, Yousefi and traits. The association between polymorphism at GH Azari (2012) detected three different conformational gene and milk yield was observed in Serra da Estrela patterns (G1, G2 and G3) in exon V of GH gene in Zel sheep by Marques et al. (2001). In Makooei sheep, a sheep breed. Though researchers did not do the significant association between polymorphism at GH sequence analysis of DNA in the mentioned patterns, gene and growth traits (weaning weight, six month weight however, their results showed that these was a significant and nine month weight) was revealed by Moradian et al. effect (p>0.05) between pattern G1 and staple strength of (2013). Moreover, Koch et al. (2010) reported that ewes wool traits. treated with GH had higher of fetal growth and development in lambs. Furthermore, in bovine, Lagziel et al. (1996, 1999) found an association between the Conclusion percentage of milk protein and polymorphism at GH gene. In conclusion, the present results confirmed polymorphism Falaki et al. (1996, 1997) revealed in Simmental and within GH gene of Egyptian sheep breeds. This poly- Holstein-Friesian cattle, the association between the morphism may be considered to be important genetic polymorphism GH-Taq1 and milk traits. Also, the marker to be used in selection programs for improving association of a polymorphism in exon 5 of GH with the wool industry. selection for milk yield was reported in Holstein-Friesian cattle by Lee et al. (1996). In contrast, Folch et al. (2001) reported that the GH Conflict of Interests treatment did not significantly have effect on the percentage of ewes in estrus and the ovulation rate. Also, The authors have not declared any conflict of interests. Yousefi et al. (2013) determined three (A, B and C) different conformational patterns in exon 5 at GH gene of Zel sheep. The frequency of pattern B (0.39) was higher Abbreviations than the pattern A (0.32) or C (0.29). However, their results revealed no significant association between PCR, Polymerase chain reaction; SSCP, single strand lambing rate and conformational patterns of GH gene. conformation polymorphisim; RFLP, restriction fragment Furthermore, DNA sequencing analysis of GH gene in length polymorphism; GH, growth hormone; Str, staple this study indicated that the sequences of PCR products strength; STL, staple length; FD, fiber diameter; CFW, are in correspondence with the sequences in the clean fleece yield; EDTA, ethylene diammine tetraacetic GenBank. SSCP analysis and sequencing allowed the acid; TBE, tris-borate EDTA. detection of a single nucleotide change for GH gene exon 5. Sequence analysis revealed a point mutation (C to T) at position 22 in the amplified fragment with accession REFERENCES number KT250511 for C allele and KT250512 for T allele. AhaniAzari M, Yousefi S, Dehanvi E (2011). 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Vol. 15(14), pp. 557-564, 6 April, 2016 DOI: 10.5897/AJB2015.14677 Article Number: DAEDAFF57898 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB
Full Length Research Paper
Effects of diets containing Cissus rotundifolia flour on lipid profile of rats and postprandial glucose levels of normoglycemic human adults
Uchenna Agatha Onyechi * and Vivienne Nkiruka Ibeanu
Department of Home Science Nutrition and Dietetics, University of Nigeria, Nsukka, Enugu State, Nigeria.
Received 25 April, 2015; Accepted 17 December, 2015
This study evaluated the effect of Cissus rotundifolia (CR), on the lipid profile of rats and postprandial blood glucose level of normoglycemic human adults. CR flour was processed using traditional Nigerian method of processing. The animal experiment involved male Sprague-Dawley rats fed for 14 days with hypercholesterolemic diet of corn oil, cholesterol, casein, mineral mix, vitamin mix, sucrose, solka floc and corn starch blended together and served as control diet (CD). The experimental diet (CRD) was CR flour added to the CD to provide 10 g total dietary fibre per 100 g but maintained similar amount of protein and fat levels as the CD. At the end of the 14 days of feeding, the rats were sacrificed and the plasma lipid levels measured. The human study involved 10 healthy subjects who fasted overnight for 12 h. Control bread meal (CB) was two whole wheat bread rolls, apricot jam, and sufficient water. Cissus rotundifolia based bread (CRB) substituted whole wheat in the test meal. Each meal contained 75 g available carbohydrate (CHO). The test meal also provided 6 g soluble non-starch polysaccharide (SNSP). The subjects were fed the CB and CRB on two separate days. Venous blood sample was taken at fasting and postprandially for 2.5 h and analyzed for plasma glucose and insulin levels. Analysis of variance and repeated measure analysis of variance (ANOVA) were used to analyze the data from the animal and human experiments, respectively. The result of the rat study showed a significant (p<0.05) reduction on the fasting plasma cholesterol and a reduction in the triglyceride levels of rats fed the CRD compared to the CD. The human study showed that CRB elicited a significant (p<0.05) decrease in plasma glucose level and insulin at 150 and 90 min, respectively. A significant reduction (p<0.005) was found in the area under the curves (AUC) with CRB compared to the CB. It was concluded that CR could be useful in modulating blood glucose levels in humans.
Key words: Cissus rotundifolia, physiological effects, lipid, glucose, rats, human.
INTRODUCTION
There has been interest in the last decade on the high fibre diet and polysaccharide gums in the dietary potential therapeutic role of a high carbohydrate (CHO), management of non-insulin dependent diabetes mellitus
*Corresponding author. E-mail: [email protected]. Tel: +2348066794814.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 558 Afr. J. Biotechnol.
(NIDDM) (Slyper, 2013). This current interest in the C. rotundifolia is a climbing or prostrate herb. The therapeutic dietary effect was generated from the leaves are glossy green, widely ovate to round with blunt epidemiological work of Trowell (1975). Studies (Jenkins toothed margins. This shrub is distributed in tropical and et al., 2002; Kaczmarczyk et al., 2012; Fujii et al., 2013) subtropical regions. CR is found throughout East Africa, have suggested a link between dietary fibre and the Zimbabwe, Mozambique, South Africa, parts of Central etiology of various chronic non-communicable diseases Africa, Egypt and the Arabian Peninsula (FAO, 1988). (NCDs). Trowell et al. (1976) noted that diabetes mellitus The young shoots of CR are used as vegetables. Their and certain metabolic diseases were rare in the minor economic importance lies in local medicinal uses developing countries where the population traditionally (FAO, 1988). The dried stem of CR is sold as food consumed diets rich in dietary fibre. However, with the condiment in the ground form. CR stem was harvested advent of western civilization and adoption of a more fresh and wet. The bark was removed by scraping. The westernized lifestyle there has been an increase in the stem was cut into small pieces and dried. The dried stem prevalence of diabetes in developing countries was crushed into smaller pieces and then ground in a (Ramachandran et al., 2012; Ram et al., 2008, Ram et coffee grinder (Moulinex blender/mill) into fine powder al., 2004). This was related to change in dietary habits and sieved. The powered CR was a yellowish brown (Burkitt and Trowell, 1976; Hall et al., 2011; Jacobs, powder. Previous work (Onyechi et al., 2007) on the 2015), economic development and market globalization nutrient content of CR flour per g/100 g dry weight (WHO/FAO, 2003). showed that CR contained high amount of available CHO World Health Organization (2000) reported that (56 g/100 g) and high available energy of 257 g/100 g. 171million people worldwide suffer from diabetes. The protein content was low (4.9 g/100 g) and contained Ramachandran et al. (2004) indicated that the greatest very little fat (0.9/100 g), and fibre (28.3 g/100 g) increase in the prevalence rate will come from the (Onyechi et al., 2007). The physicochemical properties developing countries of the world like Asia and Africa. showed that mean particle size, WBC and viscosity These authors postulated that there will be 42% increase values were 115.0, 5.85 and 5.5 dl/g respectively. in the developed countries and 170% in the developing Soluble NSP was 15.5/100 g and the starch content was countries. However, Guarigutata et al., (2014) reported 45.3 g/100 g, the sugar composition of the SNSP showed that in 2013 382 million people had diabetes; this number high levels of arabinose (8.0 g/100 g), glalactose (2.2 is expected to rise to 592 million by 2030. These authors g/100 g) and uronic acid (4.2 g/100 g) (Onyechi et al., further revealed, that most people with diabetes live in 2007). low and middle-income countries and these will The processing of CR flour and the preparation of the experience the greatest increase in diabetes cases over traditional soups is tedious and time consuming. For the next 22 years. Diabetes affects more than 6 million these reasons the use of CR flour is on the decline and Nigerians and over 1 million are blind, out of which 80% the consumption of CR vegetable soup is on the are blind from diabetes (Uduaghan, 2002). decrease in most urban areas of Nigeria. The scarcity, There are some properties of dietary fibre that are non-compliance and high cost of diabetic drugs in Nigeria related to physiological responses and food functionality makes this study of vital importance. It was therefore in the management of diabetes. These include water important to explore dietary treatment of NIDDM using binding capacity (WBC), particle size, viscosity, soluble potential indigenous foods. non-starch polysaccharide (SNSP) and nature of starch The purpose of this work was to determine the effects (Dreher, 1987; Brownlee, 2011; Ley et al., 2014). These of CR on the lipid profile of rats, as screening model. properties in both human studies (Fairchild et al., 1996; This study also evaluated the effect of CR flour on the Braaten et al., 1994; Brownlee, 2011; Dhingra et al., postprandial glucose and insulin levels in humans. This 2011) and animal studies (Johnansen et al., 1990; Ellis et work also aimed at finding a suitable food base to al., 1996) have been shown to increase viscosity of the incorporate CR flour that will be acceptable to diabetics in digesta. This is a major factor in inhibiting the rate of urban areas in Nigeria. The result of this work will digestion and absorption of available carbohydrate and determine the possibility of using CR in the prevention, consequent decrease in postprandial glucose level (Ellis management and treatment of NIDDM, as well as et al., 1996; Edward et al., 1988; Riccardi et al., 2008; diversify and increase the consumption of CR flour. Brownlee, 2011; Oputa and Chinyere, 2012). In the South Eastern Nigeria there are numerous plant MATERIALS AND METHODS foods that are used in the preparation of vegetables soups that increase the viscosity of such soups. One Metabolic study such plant food is Cissus rotundifolia (CR) which is known as “ukoho” among the Ibo tribe in the South Rat study Eastern Nigeria. CR is indigenous to Nigeria; it is used as Preparation of rat diets: Two diets were used in the rat viscous thickening agent, is easily available, cheap and experiment, the control diet (CD) and CR diet (CRD). Five kilogram processed in the homes for thickening vegetable soups. of CD was formulated using casein (New Zealand milk products Onyechi and Ibeanu 559
mix); mineral mix, vitamin mix (King’s College mix), sucrose viscosity of the flour. The CR flour was incorporated into the bread (Booker Fitch Food services), solka floc (Johnsen Jordensen and as a replacement for wheat flour. The weight of dough used was Wettre Ltd) and corn starch (Cerestar, Manchester HHIPA). These calculated such that a total of 50 g CHO was contained in two ingredients were blended together in a Hobart mixer for 15 min. bread rolls. Thus the CHO content of the flour and the other added Corn oil was heated to 80°C and the calculated amount of foodstuff were considered in arriving at the total CHO content of the cholesterol (BDH chemicals Ltd) was weighed and stirred into the bread. corn oil and mixed well to dissolve. The mixture of corn oil and cholesterol was added to the dry ingredient of casein, mineral mix, Feeding of the subjects: Ten healthy non-diabetic subjects visited vitamin mix, sucrose, solka floc and corn starch and blended for 30 the metabolic lab twice a week after an overnight fast. The subjects min until well distributed. The mixture was passed through a 1/8 were fed a control bread meal (CBM) made with only wheat flour inch mesh sieve. Homogenization of the mixture was assured by and an experimental bread made from CR flour. The meals mixing for a further 30 min in the Hobart mixer. The diet was stored consisted of two small bread rolls, 38g apricot jam (Robinson’s) and at -20°C in self-sealed freezer bags until required for the sufficient water to make a total meal weight of 400 g. The available experiment. carbohydrate portion of the meal was 75 g, the bread rolls supplied The test diet was prepared to contain Cissus flour. The amount of 50 g CHO mostly in the form of starch and the jam provided 25 g of CR flour containing 10 g dietary fibre was determined from the available CHO mostly as sucrose making a total of 75 g of available results of preliminary analysis of CR and the calculated quantity of CHO. The CRB contained 5 g of SNSP as calculated from by CR flour was added to the CD. The test diet provided approximately Englyst et al. (1992); analysis of the foods plus additional SNSP 10 g total fibre per 100 g diet while maintaining similar protein and from the wheat flour. At the start of the meal a fasting venous blood fat levels like the CD. The amount of casein and corn oil in the test sample of 10 ml was taken from each subject using EDTA diet was adjusted to maintain similar protein and fat contents with extainers. The breakfast meal was eaten within a 15 min period. the CD. Thus, the Cissus flour replaced some of the protein, fat and Postprandial blood samples were taken at 30, 60, 120 and 150 min starch in the test diet. Enough corn starch was used to make up a from the commencement of the meal. The study was approved by batch size of 5 kg diet. King’s College ethical committee.
Feeding of the rats: Twenty male Sprague-Dawley rats, supplied by A. Juck & Sons, London were used for the animal study. The Analysis of blood glucose and insulin levels rats that weighed between 71 to 87 g on arrival, were placed in a cage and fed stock diet for 2 days and ground stock diet for five Blood glucose level was measured by the glucose oxidase test days prior to the experimental period in order to acclimatize rat to using Boehringer Mannheim diagnostic kit. Blood glucose eating ground diet. After one week of acclimatization, the rats were concentration was determined after deproteinisation of the blood assigned into the two groups with a mean weight of 115 to 115 g so sample. The quantitative determination of human insulin in-vitro that rat from each litter went into an experimental group. The group was done using Boehringer Mannheim diagnostic kit based on of rats was therefore assumed to be genetically similar. The rats enzyme immunological reactions. The ES 22 combi step 22 were housed individually in cages with trays and filter paper to program, B auto machine was used for the analysis. collect spill and faeces. The rats’ food intake was recorded by providing each rat with an individual weighed pot of food and the food pots were weighed on alternate days before toping up food Statistical analysis supply and reweighing. At the end of the experiment the rats were anaesthetized and bled from the heart using heparinized needle The difference between the effect of the diets on the rats was and syringe to prevent clotting. The blood was collected and determined using analysis of variance, ANOVA, Statistical Analysis centrifuged at 2,500 rpm for 15 min before the plasma was System package, SAS Institute (1985). The level of significance separated from the cells, stored in LP3 tube at -20°C until analysis. was at p<0.05. The difference between the effect of the The plasma cholesterol was determined by enzymatic method experimental meals on the blood glucose and insulin incremental (Trinder, 1960; Roschkau et al., 1975; Siedel et al., 1981) supplied values were determined by repeated measures ANOVA with by Boehringer - Mannheim (Boehringer CHOD/PAP kit method). statistical package (Statistical Analysis System package, SAS The principal is based on the hydrolysis of cholesterol ester to free Institute (1985). Integrated glucose and insulin increments were cholesterol and fatty acids by cholesterol ester hydrolase. Free estimated by calculation of area under the curve (AUC; trapezoid cholesterol is oxidized by reacting with oxygen in the presence of rule). cholesterol oxidase and hydrogen peroxide is formed which reacts with a chromophore to produce a pink colour The absorbance of the resulting solution was read in a spectrophotometer (UV/VIS RESULTS spectrophotometer, Philip instrument, UK), against a blank at 500nm. The plasma triglyceride of the rats was analyzed using the Rat study enzymatic method described by Tiffany et al., 1974. The Boehringer Mannheim kit method was used for this analysis and Table 1 gives a description of the composition of the absorbance of the sample was read against a blank at 500nm control diet and experimental diet which was essentially wavelength in a spectrophotometer. the same expect the addition of the experimental food CR
flour. In Table 2 the result of the nutrient analysis of the Human study rats diet is shown. The nutrients were comparable expect for the fibre in the CRD, which is due to the additional Preparation of the experimental bread: The bread rolls were fibre from the CR flour. prepared using the Chorleywood bread process (Apling and Ellis, Table 3 shows the effect of CD and CRD diets on the 1982). The brown wheat flour used was Ploughman’s brown flour (Sovereign, Allied Mills, London EC3R 7PE). The hydrogenated fasting plasma cholesterol and triglyceride levels vegetable fat was the flora brand (Unilever, UK). Each batch of (mmol/L) of rats. The result showed that mean plasma bread dough contained variable amounts of water depending on the cholesterol levels of rats fed CRD was significantly lower 560 Afr. J. Biotechnol.
Table 1. Composition of control diet (CD) and diet containing CR (CRD) fed rats.
Quantity of ingredients (g/1000 g) diet Ingredients CD CRD Casein 150 133 Fat (corn oil) 100 100 Vitamins 20 20 Minerals 40 40 Sucrose 100 100 Cholesterol 10 10 Solka floc 50 50 Food samples - 354 Corn starch 530 193
Table 2. Nutrient analysis of the control diet (CD) and diet containing CR (CRD) fed to rats.
Nutrients CD CRD Moisture (%) 6.11 5.89 Fat (%) 10.00 10.1 Protein (%) 15 15 Ash (%) 4.8 5.1 Available CHO (%) 58.0 45.0 Fibre by difference (%) 6.1 19.0 Total CHO 64.1 63.6 406.0 404.0
Table 3. Fasting plasma cholesterol and triglyceride levels mean (mmo/L) of rats fed control diet (CD) and CR test diet (CRD).
Diet Plasma cholesterol level (mmol/L) Plasma triglyceride levels (mmol/L) CD 3.97±0.17 a 0.80±0.07 a CRD 3.32±0.09 a 0.63±0.11b
Values are mean ± sem. (n=10); Column values with same superscript are significantly different from each other at p<0.05.
than those fed the CD at p<0.05. The result of the plasma the CRB showed that there was no significant difference triglyceride levels of rats fed the CD and CRD showed no but there was apparent decrease. The difference significant reduction. However CRD showed a lowering between the bread meals was analysed at the time effect. intervals, the CRB showed a significant difference at 150 min. The effect of the bread meals on the incremental insulin levels is shown in Figure 2. Comparison of the Human study mean incremental insulin rise after the consumption of the CB and CRB showed no significant difference In Table 4 the food ingredients used for the production of between the two bread meals, however there was the CB and CRB were shown and the nutrient apparent reduction with CRB. The difference between composition of the meals were shown in Table 5. Figure the bread meals was analysed at the time intervals, the 1 shows the effect of the bread meals on the incremental CRB showed a significant difference at 90 min. A blood glucose levels. The comparison of the mean significant reduction (p<0.005) was found in the area incremental glucose rise after the consumption of CB with under the curves (AUC) of CRB compared to the CB. Onyechi and Ibeanu 561
Table 4. Food ingredients used for the preparation of control bread (CB) and test bread containing CR (CRB).
Quantity of ingredients (g/1000 g flour) Ingredient CB CRB Brown flour 1000 550 Salt 18 18 Experimental foods - 450* Fat(hydrogenated) 7 7 Improver 100 100 Fresh yeast 25 25 Water 675 750
*Equivalent to 63 g soluble fibre (Englyst analysis).
Table 5. Nutrient composition of bread meals fed human non-diabetic adults (n=10).
Type of bread Amount (g) Protein (g) Fat (g) Available CHO (g) Total SNSP Control bread 136 10.2 1.9 70.0 1.8 Cissus bread 148 7.9 1.7 70.1 6.1
2 1.8
1.6 CB 1.4 CRB 1.2 1 0.8 0.6 0.4 0.2
Incremental plasma glucose level (mmol/L) level glucose plasma Incremental 0 30 60 90 120 150
Time (min)
Figure 1. Mean incremental plasma glucose levels (mmol/L) of healthy subjects fed control bread and CRB. n =10.
DISCUSSION reduction (p<0.5) on plasma glucose (Figure 1) and insulin (Figuere 2) at 150 and 90 min, respectively. A The animal study showed a significant decrease (p<0.05) significant reduction (p<0.005) was found in the area in the plasma cholesterol level of rats fed the CRD under the curves (AUC) for glucose and insulin with the compared to the CD as shown in Table 3. However there consumption of CRB compared to the CB. C. rotundifolia was no significant reduction in the plasma triglyceride is the stem of a shrub and limited research work has levels of rats fed CRD compared to the CD but there was been done to examine such materials as CR. A material apparent reduction with CRD (Table 3). The result of the which may be similar to CR is konjac mannan, which is human study shows that CRB elicited a significant prepared from fresh roots and has shown to reduce 562 Afr. J. Biotechnol.
45 40 35 CB 30 CRB 25 20 15 10 5 0
-5 30 60 90 120 150 Incremmental plasma insulin level (mU/L) level insulin plasma Incremmental Time (min)
Figure 2. Mean incremental plasma insulin levels (mU/L) of subjects fed control bread and CRB. n=10.
postprandial plasma glucose and insulin concentration in dietary propionate lowered serum and liver cholesterol. humans (Ebihara et al., 1981). These authors indicated The authors suggested that this reduction is due to that beneficial effects of konjac mannan which contain propionate inhibiting hepatic cholesterol synthesis. This viscous soluble NSP just as CR, were related to delayed same mechanism is attributable in the rats fed CRD since stomach emptying, modified responses in gastrointestinal CR flour contain SNSP hence the decrease in cholesterol hormones and delayed glucose diffusion. Delayed gastric levels in the rats. It is also possible that CR which is high emptying was a major factor in reducing plasma glucose in starch produce SCFA from both the SNSP and the and insulin concentration as well as decreased starch, indicating a high level of propionic acid with intraluminal glucose diffusion (Brownlee, 2011). CR has hypocholesterolemic effect (Chen et al., 1984; Brownlee, some similarity with konjac mannan, both contain soluble 2011). NSP, although one is described as a root and the other a Water binding capacity (WBC) is the maximum amount stem, they are both “woody” parts of plants which contain of water that can be taken up per unit weight of dry food viscous soluble NSP. The result of this study shows that material in the presence of excess water. It is the ability CR flour has the same physiological effect on human and of fibre to hold water under specific conditions. Onyechi animals just as konjac mannan due to the similarity their et al. (2007) showed that CR flour had a WBC of 5.58. physico-chemical properties (Nichenametla et al., 2014). Studies have also shown that NSPs like CR with high CR had a viscosity of 5.5 dl/g, and a SNSP content of WBC prolong gastric emptying (Bueno et al., 1981) 14.6 g/100 g (Onyechi et al., 2007). Jenkins et al. (1978) hence decreased postprandial blood glucose levels in showed a positive correlation between mean peak rise in human. blood glucose and gum viscosity of a wide variety of CR has a mean particle size of 115 µm (Onyechi et al., viscous NSPs. The authors postulated that the action of 2007). It has been shown that particle size or the texture these viscous materials maybe two fold, delay in gastric of food plays an important role in glycaemic response. emptying and delay in glucose absorption from the small Food of coarse consistency produced smaller glycaemic intestine. Viscous gels like CR entrap glucose and slowly responses than finely milled food grains which break the release it for absorption. This effect can explain the cell walls, producing a quicker access of amylase to the reduction in the postprandial blood glucose concentration starch flour (O’Dea et al., 1980). Particle size is property in humans fed CRB. of dietary fibre that helps in modulating the postprandial The effect of CRD on the lipid profile of experimental blood glucose level. This particular property is attributable rats is attributable to increased fecal bile acid excretion. to CR flour; hence the positive result obtained in the Cummings (1981) indicated that the loss of these postprandial glucose and insulin levels in the human cholesterol containing compounds has study and the reduction in the lipid profile of rats. hypocholesterolemic effects. Studies have shown that CR flour contained a high level of starch (45.3 g/100 g) SNSP are almost completely fermented by colonic (Onyechi et al., 2007). Studies have shown a reasonable bacteria to produce short chain fatty acids (SCFA) like correlation between glycaemic index, and in vitro acetate, propionate and butyrate (Cummings, 1981). digestibility of starchy foods (Holm et al., 1988; Dhingra Chen et al. (1984) in their study with rats showed that et al., 2011; Brownlee, 2011; Bodinham et al., 2014). Onyechi and Ibeanu 563
Crapo et al. (1971) showed that the glycaemic and D, Mumpleby AM, Robertson MD (2014). Efficacy of increased insulin response in humans were not only related to the resistant starch consumption in human Type 2 diabetes. Endocr. Connect. 3(2):75-84. dietary fibre content of the foods but also to the difference Braaten JT, Scott FW, Wood PJ, Reidel KD, Poste LM, Collins MW in the digestibility of different starches (Coulston et al., (1994). High β-glucan oat bran and oat gum reduce postprandial 1980; Jenkins et al., 2002, Allen et al., 2011). Goddard et blood glucose and insulin in subjects with and without type 2 al. (1984) showed that amylose/amylopectin ratio is a diabeteis. Diabet. Med. 11:312-318. Brownlee IA (2011). The physiological roles of dietary fibre. Food contributory factor to low glycaemic index. Foods that are Hydrocoll. 25(2):238-250. known to have high levels of amylose in their starch Bueno L, Praddante F, Fioramonti J, Ruckebusch Y (1981). Effect of granules are more slowly digested compared to dietary fibre on gastrointestinal motility and jejuna transit time in carbohydrate foods that contain less amylose and more dogs. Gastronterology 80:701-707. Burkitt DP, Trowell HC (1976). Refined carbohydrates and diseases, amylopectin (Goddard et al., 1984; Benhall et al., 2006). some implications of dietary fibre. London, UK: Academic press Inc. This is due to the fact that amylopectin has larger surface Chen WJL, Anderson JW, Jenkins D (1984). Propionate may mediate area per molecule than amylose. In addition amylose unit the hypocholesterolemic effects of certain soluble plant fibres in are more bound to each other by hydrogen bonds making cholesterol fed rats. Proc. Soc. Exp. Biol. Med. 175:215-218. Coulston A, Greenfield M, Kramer F, Tokey T, Reaven G (1980). Effect them less available for amylolitic attack (Schooch and of source of dietary carbohydrate on plasma glucose and insulin Maywald, 1976). However, the structure of the starch in responses to test meals in normal subjects. Am. J. Clin. Nutr. CR flour has not been studied so the 33:1279-1282. amylose/amylopectin ratio is not known. However, CR Crapo P, Reaven G, Olefsky J (1971). Post-prandial plasma glucose and insulin responses to different complex carbohydrates. Diabetes starch has attributes that makes it resistant to digestion, 26:117-183. as indicated in the postprandial glucose and insulin Cummings JH (1981). Dietary fibre. Br. Med. Bull. 37:65-70. human subjects and decreased plasma lipids in the rat Dhingra D, Micheal M, Rajput H, Patil RT (2012). Dietary fibre in foods: study result (Bodinham et al., 2014). a review. J. Food Sci. Technol. 49(3):255-266. Dreher ML (1987). Handbook of dietary fibre. O.R Fennema, G.W. Sanderson, P. Walstmara, M. Karel, S.R. Tannenboum, J.R. Whitaker eds. New York: Marcel Dekker Inc. Conclusion Ebihara K, Masuhara R, Kiriyama S (1981). 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