Stigma -5, 22, -Diene-3-O-β-D-Glucopyranoside: A new antimicrobial glycoside from Tetrapleura tetraptera

Indigenous medical practices have been the subject of much attention in the literature of various disciplines to date but the specificity, mode of action and clinical efficacy of most traditional plants have not been established in a manner consistent with standards of modern pharmacognosy and pharmacology. In order to find antimicrobial lead compounds and to provide scientific validation for the use of Tetrapleura tetraptera (Schum.& Thonn.) in traditional medicine . Extensive chromatographic separation of stem bark and leaf extracts led to the isolation of three distinct compounds. Anti-microbial spectrum of these obtained compounds revealed that stigma-5, 22, diene-3-O-β-D-glucopyranoside showed activity against Staphylococcus aureus (32 mm), Streptococcus pyrogenes (30 mm) and Candida tropicalis (30 mm). It is therefore recommended for further work for a possible development of new drugs.


Introduction
Traditional medicine practices have remained a module of health care system of many societies in spite of the accessibility of well-established alternatives [1]. It is our socio-economic and socio-cultural legacy as well as the earliest choice healthcare treatment for at least 80% of Africans who suffer from high fever and common ailments [2]. This is due to the fact that traditional medicine is the most inexpensive and available system of healthcare [3]. Therefore, many people in the developing countries still rely on traditional healing practices and medicinal plants for their vital healthcare needs [4]. In industrialized countries, adaptations of traditional medicine are termed "Complementary" or "Alternative" Medicine.
The World Health Organization (WHO) estimates that in several African countries traditional birth attendants assist in the majority of births [5]. In Ghana, Mali, Nigeria and Zambia, the first line of treatment for 60% of children with high fever resulting from malaria is the use of herbal medicine at home [6]. The World Health Organization (WHO) encourages the inclusion of herbal medicine of proven safety and efficacy in the healthcare programmes of developing countries [7]. The extent of sensitization and mobilization by the WHO has encouraged some African countries to initiate serious development on traditional medicine [8]. Over the past years, plants have become an indispensable source of food and medicine. To a large extent, most people depend greatly on medicinal plants as an important source of remedy and treatment for most casual and life-threatening diseases. As a result, there is a growing demand all over the world for these medicinal plants. Aside from tackling diseases, people are resorting more to these medicinal plants as a means of reducing the use of chemical (orthodox) medicines that could potentially be detrimental to human health. Interestingly, most of these plants are used in our everyday food preparations as herbs, spices, seasonings and preservatives. But the truth is that we often consume most of these essential medicinal plants in the form of spices without even acknowledging what our bodies gain from them. Medicinal plants have contributed significantly as starting points for the development of modern drugs, as evidenced by taxol in cancer and artemisinin in malaria [9].This may be attributed to their chemical diversity, biomedical specificity, possession of a greater number of chiral centres than in synthetic or combinatorial libraries, and evolutionary pressures to create biologically active compounds by interactions with different proteins and biological targets [10]. Plants therefore, represent potential sources of new drugs acting through novel mechanisms in the search for new and more potent bioactive agents that target a variety of ailments including tuberculosis. The medicinal properties of plants have not been sufficiently harnessed. The difficulty encountered with alternative medicine has been that of reliable documentation of known traditional herbal medicine since uses vary from tribe to tribe.
Indigenous medical practices have been the subject of much attention in the literature of various disciplines to date but the specificity, mode of action and clinical efficacy of most traditional plants have not been established in a manner consistent with standards of modern pharmacognosy and pharmacology. The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are flavonoids, steroids, saponins, tannins, alkaloids and phenolic compounds [9]. Many of these indigenous plants are used as spices and food plants. Therefore, with the increased demand for organic materials that serve as food additives, constituents of functional foods, nutraceuticals and prevention of plant diseases, it is imperative to examine the applicability and benefits of indigenous plant materials to mankind using modern analytical methods [11]. In Nigeria, therapeutic information for a lot of such plants is based on folklore and one of such plants suggested by folklore for the treatment of different pathogens that trigger malaria infections is Tetrapleura tetraptera, (Figure 1) commonly called "Aidan tree" in South-west region of Nigeria [12]. As part of our investigation on some medicinal plants in Nigeria, the anti-microbial activity displayed by isolated compounds from T. tetraptera is being reported for the first time from this plant.

Figure 1
Picture of Tetrapleura tetraptera leaves and fruits.

General experimental procedures
The infrared (IR) spectra were obtained on a Perkin Elmer Spectrum 100 Fourier transform infrared spectrophotometer (FT-IR) equipped with universal attenuated total reflectance (ATR) sampling accessory (Perkin Elmer, Waltham, USA). 1 H, 13 C and 2D nuclear magnetic resonance (NMR) spectra were obtained using deuterated dimethyl sulfoxide (DMSO) or chloroform (CDCl3) at room temperature on a Bruker Avance III 400 spectrometer (Bruker, Rheinstetten, Germany). High resolution mass spectra (HRMS) were obtained on a Waters Micromass LCT Premier Time of Flight-Mass Spectromerty instrument (Waters, Massachusetts, USA). Column chromatography was done using Merck silica gel 60 (0.040-0.063 mm) and Merck 20 cm × 20 cm silica gel 60 F254 aluminium sheets were used for thin-layer chromatography (TLC). The TLC plates were viewed under ultraviolet (UV) (254 and 366 nm) lamp before further visualization using a stain made of 10% sulfuric acid in methanol (MeOH) solution followed by heating. Organic solvents of analytical grade and other chemicals used were purchased from Merck (Darmstadt, Germany) or Sigma (St. Louis, USA) chemical companies.

Plant Material
The leaves and stem bark of Tetrapleura tetraptera ( Figure 1) were collected in fresh condition from Owo region of Ondo state, Nigeria. Taxonomical identification was done at the Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria and herbarium number (FHI110372) was deposited at the Institute.

Extraction of plant material
Each ground plant sample (1000 g) was exhaustively extracted using Soxhlet extraction method with 95 % ethanol as the solvent [13]. The extracts were concentrated at 40˚C using a rotary evaporator and later air-dried to give dried crude extracts.

Fractionation of crude extracts
The crude residues of stem bark and leaves were fractionated into acidic, basic, polar neutral and non-polar neutral using a modified bio-assay guided method [14]. A flowchart of the intricate steps is depicted below (Figure 1

Chromatographic Separation of fractions
The column was packed with fine silica gel (Kieselgel 60), a column having 40 cm length and 3 cm in diameter was packed with 150 g of silica gel up to a height of 23 cm under reduced pressure. The column was washed with n-hexane to facilitate compact packing. The neutral polar fraction (13.0 g) of the stem bark loaded onto a silica gel column using wet method and eluted with hexane : ethyl acetate: methanol (100:0-100:10 v/v) gradient to give 68 fractions of 100 ml each. Fractions with similar TLC profiles were combined. Fractions 19-20 were combined and purified in hexane to give compound A, a white needle-like crystal (18 mg) and fractions 52-53 were filtered out from 100 % ethyl acetate to obtain a white solid crystal compound B (19 mg). Fractions 59-60 were also purified using 100 % ethyl acetate and filtered to give a white cotton-wool like solid compound C (42 mg).The neutral non-polar fraction (15 g) of the leaf was subjected to column chromatography using hexane: ethyl acetate: methanol (100:0-100:20, v/v) gradient to give 60 fractions of 100 ml. Fractions 29-31 were combined to give a dark green amorphous solid compound D (15 mg); and neutral polar fraction of the leaf (20.0 g) was also subjected to column chromatography using the same solvent system, 52 fractions of 100 ml were collected. Fractions 8-9 were washed with acetone, dried and to give a shiny white compound E (19 mg).

Antimicrobial Screening
All the media were purchased from Sigma-Aldrich and were prepared in accordance with the manufacturer's instructions. The clinical bacterial and fungal isolates were obtained from Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, Kaduna State, Nigeria. The identities of all isolates used were confirmed using standard biochemical test [26]. Agar diffusion method adopted from EUCAST [27] was employed. The minimum inhibitory concentration (MIC) was determined on the test isolates and was done by broth dilution method [28]. Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were evaluated by plating the bacterial suspensions from individual well at the beginning and at the end of the experiments on Muller Hinton agar medium for estimation of MBC [28]. The MBC / MFC values were the plates with lowest concentrations of the isolates without colony growth.

Spectroscopic characterization
Chromatographic separation of neutral polar stem bark and non-polar leaf fractions gave five compounds which were subjected to different spectroscopic analyses to elucidate the structures of the isolated compounds.

Discussion
The five compounds (1)(2)(3)(4)(5) were isolated from the stem bark and leaves of Tetrapleura tetraptera. The compounds belonging to a sterol, a saponin, mono-N-acetyl glycoside, a straight chain fatty alcohol and a natural pigment were identified based on their 1 H and 13 C-NMR spectra and 2D NMR spectra which were compared to values in literature. This is the first time these compounds were isolated from T. tetraptera stem bark (except a mono-N-acetyl glycoside) and leaf though they are known compounds.
The compound A is a white needle-like crystal with melting point 162-168 0 C which gave positive Salkowski and Lieberman-Burchard test for steroid (Figures 3-9). The FTIR (Fourier Transform Infra-red) spectrum exhibits characteristic absorption band at 3336 cm -1 which is the O-H stretching. Absorptions at 2918 and 2848 cm -1 are due to aliphatic C-H stretching. The absorptions at 1638 and 1052 cm -1 are as a result of C=C and C-O stretching. These assignments with reported values were all in agreements with other reported studies [15]; [16]. The 1 H spectrum showed the presence of six methyl groups of a stigmastane carbon skeleton, identical to stigmasterol which appeared at δH 0.81, 0.83, 0.85, 0.88, 1.08 and 1.65 ppm. The de-shielding effect of protons at δH 3.53 ppm (1H, m, H-3) indicated, they were bonded to oxygenated tertiary carbon. The 13 C spectrum displayed 29 carbons signals including six methyls. The correlation of carbons to their respective hydrogens was found by using HSQC (Heteronuclear Single Quantum Correlation) spectrum and the assignments were supported by H-C correlation based on HMBC (Heteronuclear Multiple Bond Correlation) spectrum. Signals at 140.75, 121.70 ppm are for C-5 and C-6 double bond while 71.80 ppm for C-3 hydroxyl group, 138.31 ppm for C-22 and 129.28 for C-23 respectively. Therefore C-5, C-6, C-22 and C-23 are alkene carbons. If we compare DEPT (Distortionless Enhancement by Polarization Transfer) 90 and 135 experiments, we confirmed that this compound was having six methyl, nine methylene, eleven methine and three quaternary carbon groups. Similarity of the above spectral data (Figures 3-8) to those of published data [17]; [18] suggested that it is stigmasterol, 2.
Compound B gave a positive Liebermann-Burchard test and IR spectrum exhibited strong bands at 3383 and 1050 cm -1 characteristic of a glycoside ( Figure 10). The calculated m/z suggested to be 574.42 but due to the loss of glucose from the molecular ion peak, gave a fragment at m/z 396. .69, one methylene resonance at δ 61.04 were due to C-2 ' , C-3', C-4', C-5' and C-6' respectively of the β-Dglucopyranoside. The chemical shift of the anomeric carbon at 100.76 (C-1') as well as the other sugar carbon chemical shifts were in agreement with α-configuration and with a pyranose form [19]. The olefinic resonances at δ 121. 13, 137.99, and 128.79 corresponded to the C-6, C-22 and C-23 methine carbons and a signal at 140.42 corresponded to the C-5 quaternary carbon of the sterol moiety. The COSY and relayed COSY experiments showed that the anomeric doublet at δ 4.22 belonged to one glucose residue. In the COSY spectra all coupling constants were large and conformed to the equatorial proton of one β -D-glucopyranose residue. If one has a pure stereoisomer, the presence of one peak as a doublet between δ 4.0 and 4.6 with J around 8 Hz will point to β-isomer. While a doublet between δ 4.6 and 5.2 (comparatively de-shielded) with smaller J around 4 Hz, the presence of α-isomer will be indicated. The HMBC showed correlation between H-1' and C-3 (H-1'→C-3) and based on the above spectral data (Figures 9-14) which were in complete concurrence with the literature [8]; [20] this compound was identified as stigmast -5, 22-diene-3-O-β-Dglucopyranoside, 3.
Compound D appears to be a natural pigment called pheophytin a and was obtained from the non-polar fraction of the leaf of T. tetraptera. Pheophytins are the degraded products of chlorophylls. During metabolite extraction the chlorophylls may lose their magnesium ions and become pheophytins. The signals of part of phytal fragments (-O-CH2-CH=C (CH3)-) of chlorophylls and other parts of pheophytins probably appeared at δ 9.47, 9.33, 8.537.98, 3.37 and 3.18 [23]. The ratio of chlorophylls a and b was determined as 3:1 by integration of singlet signals at δ 9.37 and 9.55. Other minor signals in the range of δ 11-7 may have appeared due to oxidized products of chlorophylls. The presence of two signals in the up field region (δ -1.46 and -1.65) of the spectrum of leaves was characteristic of N-H group of the porphyrins (Figures 21-26) [23]. The identification of this compound as pheophytin a, 4 was achieved by comparing the 1 H-NMR, 13 C-NMR, HMBC, HSQC, COSY and NOESY spectroscopic data with those in literature [5]; [24].
The antimicrobial efficiency of selected isolates was investigated under the same parameters as the crude and bioassay guided fractions (Tables 1, 2

Conclusion
Isolation of three new active compounds and a natural pigment were compared with that of literature and confirmed to be stigmasterol, Stimast -5, 22-diene-3-O-β-D-glucopyranoside, n-tetraeicosanol and Pheophytin which are known compounds that have very good medicinal potentials. More compounds should be isolated from other bioassay guided fractions, subjected to biological and pharmacological studies to investigate the medicinal potential of Tetrapleura tetraptera. Also, derivatives of the isolated compounds should be synthesized to improve on its activity.