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Inhibitory activities of selected Sudanese medicinal plants on Porphyromonas gingivalis and matrix metalloproteinase-9 and isolation of bioactive compounds from Combretum hartmannianum (Schweinf) bark
© The Author(s). 2017
Received: 19 August 2016
Accepted: 8 April 2017
Published: 20 April 2017
Periodontal diseases are one of the major health problems and among the most important preventable global infectious diseases. Porphyromonas gingivalis is an anaerobic Gram-negative bacterium which has been strongly implicated in the etiology of periodontitis. Additionally, matrix metalloproteinases-9 (MMP-9) is an important factor contributing to periodontal tissue destruction by a variety of mechanisms. The purpose of this study was to evaluate the selected Sudanese medicinal plants against P. gingivalis bacteria and their inhibitory activities on MMP-9.
Sixty two methanolic and 50% ethanolic extracts from 24 plants species were tested for antibacterial activity against P. gingivalis using microplate dilution assay method to determine the minimum inhibitory concentration (MIC). The inhibitory activity of seven methanol extracts selected from the 62 extracts against MMP-9 was determined by Colorimetric Drug Discovery Kit. In search of bioactive lead compounds, Combretum hartmannianum bark which was found to be within the most active plant extracts was subjected to various chromatographic (medium pressure liquid chromatography, column chromatography on a Sephadex LH-20, preparative high performance liquid chromatography) and spectroscopic methods (liquid chromatography-mass spectrometry, Nuclear Magnetic Resonance (NMR)) to isolate and characterize flavogalonic acid dilactone and terchebulin as bioactive compounds.
About 80% of the crude extracts provided a MIC value ≤4 mg/ml against bacteria. The extracts which revealed the highest potency were: methanolic extracts of Terminalia laxiflora (wood; MIC = 0.25 mg/ml) followed by Acacia totrtilis (bark), Ambrosia maritima (aerial part), Argemone mexicana (seed), C. hartmannianum (bark), Terminalia brownii (wood) and 50% ethanolic extract of T. brownii (bark) with MIC values of 0.5 mg/ml. T. laxiflora (wood) and C. hartmannianum (bark) which belong to combretaceae family showed an inhibitory activity over 50% at the concentration of 10 μg/ml against MMP-9. Additionally, MMP-9 was significantly inhibited by terchebulin with IC50 value of 6.7 μM.
To the best of our knowledge, flavogalonic acid dilactone and terchebulin were isolated from C. hartmannianium bark for the first time in this study. Because of terchebulin and some crude extracts acting on P. gingivalis bacteria and MMP-9 enzyme that would make them promising natural preference for preventing and treating periodontal diseases.
Periodontal diseases are multifactorial infections caused by a specific group of Gram-negative anaerobic bacteria leading to destruction of the tooth-supporting tissue including the alveolar bone and the periodontal ligament. Two major factors contributed to the pathogenesis of periodontitis are namely periodontopathogens which cause direct damage to periodontal tissue through the secretion of toxic products, and the host response to periodontopathogens which results in the release of inflammatory mediators (proinflammatory cytokines, matrix metalloproteinases (MMPs) and prostanoids) .
Porphyromonas gingivalis, a Gram-negative, black pigmented and an anaerobic bacterium, has been strongly implicated in the etiology of some types of periodontitis including chronic adult periodontitis [2, 3]. It is a major periodontal pathogen that possesses multiple virulence factors including gingipains, lipopolysaccharides and can trigger host cells to release inflammatory cytokines and MMPs . Previous studies showed that MMP-9 secretions were unregulated by P. gingivalis supernatant in periodontal ligament fibroblasts, pulp fibroblasts and osteosarcoma cells [5–7].
Selected Sudanese medicinal plant species used in traditional medicine
Calotropis procera (Aiton) Dryand
Arestolochia bracteolate Lam.
Xanthium brasilicum Vell.
Vernonia amygdalina Delile
Fever, gastro-intestinal disease “GID” .
Adanosonia digitata L.
The fruits are used as a cold beverage, added to yoghurt for treatment of diarrhea and amoebic dysentery .
Terminalia laxiflora Engl.
Terminalia brownii Fresen
Combretum hartmannianum (Schweinf)
Ambrosia maritima L.
The herbs are used in treatment of urinary tract infections and elimination of kidney stones, whereas the leaves are used as anti-diabetic and anti-hypertensive .
Euphorbia hirta L.
Decoction of plant is use in asthma and bronchitis .
Ricinus communis L.
Acacia seyal var. fistula (Schweinf.)
Fumigation, rheumatic pain .
Acacia seyal var. seyal Del.
Anti-rheumatic, mouth detergent .
Acacia tortilis (Forssk.) Hayne
Treat skin infection, allergic dermatomes .
Cassia acutifolia Delile
Parkinsonia aculeata L.
Antipyretic, anti- diabetics .
Senna italica Mill.
Intestinal complications, haemomorphoids, circulatory system problems, calculi in the urinary system, sexually transmitted diseases .
Khaya senegalensis (Desv) A. Juss
Anti-malarial, against hepatic inflammation, sinusitis, skin diseases, GID, trachoma .
Polygonum glabrum Willd
Anthelminthic, antimalarial .
Argemone mexicana L.
Venereal diseases .
Solanum dubium Fresen
The whole plant and fruits are pulped and applied to wounds and skin tumors as a dressing .
Salvadora persica L.
Tamarix nilotica (Ehrenb.)Bunge
Tarft al nil
Febrile, colds .
Tribulus terrestri L.
Demulcent, renal nephritis .
Combretum hartmannianum a shrub up to 4 m; as a tree under favorable conditions 10 m high. The plant is widespread throughout the Sahel belt from Senegal to Cameroon, and eastwards to the Sudan . Leaves, fruits and stem bark extracts of C. hartmannianum showed activity against Gram-positive bacteria, E. coli (Gram-negative); and have also been reported to exhibit anti-inflammatory activity [13, 14].
Hence, the purpose of this study was firstly to investigate the antibacterial activity against P. gingivalis bacteria of 62 methanolic and 50% ethanolic extracts from 24 selected Sudanese medicinal plants species. Secondly, from these 62 extracts; seven methanol extracts (Terminalia laxiflora, Tamarix nilotica, Khaya senegalensis, Acacia seyal var. fistula, Acacia seyal var. seyal, C. hartmannianum and Terminalia brownii) were selected to examine their inhibitory activities against MMP-9 enzyme. Additionally methanolic extract of C. hartmannianum bark that demonstrated good combined activities were subjected for further fractionation in order to identify the active compounds responsible for the biological activities.
Twenty four different plant species were collected from Khartoum and Elgadarif States, Sudan, identified and authenticated by Dr. Ashraf Mohamed from the Faculty of Forestry, Mrs. Hamza Tag EL-Sir Herbarium Curator. Voucher specimens (Table 3) were deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum.
Preparation of plant extracts
Different plant parts (Table 3) were dried under shade and then grounded before they were subjected to cold maceration with methanol or 50% ethanol. The plant powder was macerated with a gentle shaking for 12 h three times in solvents in side stoppered flasks at room temperature. The extracted solvents were filtrated and evaporated under reduced pressure using a rotatory evaporator, and the concentrated 50% ethanol extracts were then dried with a freeze dryer, resulting in 62 crude extracts and stored at 4 °C until use. In order to prepare stock solution, extracts were dissolved in 100% dimethyl sulfoxide (DMSO). Further serial dilution of the stock was performed to obtain a range of desired concentration of the extracts.
Fractionation, purification and isolation of Combretum hartmannianum bark
Five grams of C. hartmannianum bark methanolic extract was subjected to fractionation by medium pressure liquid chromatography (MPLC) using ODS column (YMC-DispoPack AT ODS-25:120 g). The column was conditioned with the first eluent used for separation for 30 min with flow rate 0.5 ml/min. MPLC separation was performed by using a chromatography pump (540 Yamazen, Japan), UV detector at 280 nm wavelength (UV-10 V Yamazen, Japan) and a fraction collector (SF-2120, Advantec Tokyo Ltd., Japan). Elution with H2O/MeOH 95/5, 20/80 and absolute methanol resulted in three fractions (F1, F2 and F3). Fraction one (F1), which demonstrated a good inhibitory activity against bacteria and enzyme, was subjected to column chromatography on a Sephadex LH-20 eluted with methanol (90–20%) in water, and finally washed with 70% acetone to give five sub-fractions. Separation of these sub-fractions mainly, (F1–1, F1–2 and F1–3) were performed by using preparative high performance liquid chromatography (HPLC) with reversed phase Inertsil ODS-3 column (GL Sciences Inc. 10 mm i.d. × 250 mm) monitored at 280 nm. The solvent system used was as follows: a gradient program for 60 min from 10 to 100% methanol in water with 0.05% TFA at a flow rate 5 ml/min .
Flavogallonic acid dilactone
1H (ppm) JH,H (Hz)
1H (ppm) 
13C (ppm) 
1H (ppm) JH,H (Hz)
1H (ppm) 
6.56 (s, H)
6.63 (s, H)
6.79 (s, H)
6.80 (s, H)
7.48 (s, H)
7.58 (s, H)
6.37 (s, H)
6.37 (s, H)
6.42 (s, H)
6.39 (s, H)
5.23 (d, J = 2.8 Hz)
5.32 (d, J = 4 Hz)
4.98 (dd, J = 3.5, 9.7 Hz)
4.88 (dd, J = 3.9 Hz)
5.64 (t, J = 9.6 Hz)
5.59 (t, J = 8 Hz)
4.78 (t, J = 11.0 Hz)
4.21 (t, J = 10.3 Hz)
3.04 (t, J = 11.6 Hz) 4.48 (t, J = 8.9 Hz)
3.10 (d, J = 12 Hz)
Determination of minimum inhibitory concentration (MIC)
MIC was determined by the broth dilution method according to Iwaki et al. . Prophyromonas gingivalis ATTC 33277 was cultured in a Brain-Heart Infusion broth supplemented with 0.5 μg/ml vitamin K and 5 μg/ml hemin. The crude extracts and pure compounds were tested for antibacterial activity in sterile 96-well plates. The inoculums were prepared by diluting the broth culture to approximately 108 cell/ml. To each well; 100 μl of microbial inoculums were added and followed by addition of media to achieve a final volume of 200 μl. The tested extracts or isolated compounds were prepared in a concentration range of 4000–31.3 μg/ml using a two-fold dilution method. The experiments were performed in triplicate. Chlorhexidine was included in the assays as positive control. The cultures were incubated for 72 h at 37 °C under anaerobic conditions. Microbial growth was indicated after the addition of 50 μl of (0.2 mg/ml) p-iodonitrotetrazolium violet (INT) to the cultures and incubated at 37 °C for 2 h. The MIC was defined as the lowest concentration that inhibited the color change of INT .
Measurement of collagenase activity
VI: reaction velocity of (sample or inhibitor).
VC: reaction velocity of control.
The percentage and IC50 values of MMP-9 inhibitory activities were expressed as the mean value. The significant differences between extracts or isolated compounds were assessed by one-way analysis of variance (ANOVA) followed by pair wise comparison of the means using Tukey’s multiple comparison test. Values were determined to be significant when p was less than 0.05 (p < 0.05).
Results and discussion
In this study methanol and 50% ethanol were chosen as solvent for extraction. As shown in the previous studies, nearly all of the identified components from plants active against microorganisms and enzyme may be related to the polyphenolic content of the plant extract, so the initial screenings of plants can be done by using crude aqueous or alcohol extraction [21, 22].
Evaluation of MIC activity of plant extracts against P.gingivalis
Minimum inhibitory concentration (MIC) activities of selected Sudanese medicinal plants against P. gingivalis
A. bracteolate Lam.
A. digitata L.
A. maritima L.
A. mexicana L.
A. tortilis (Forssk.) Hayne
A.seyal var. fistula (Schweinf.)
A.seyal var. seyal Del.
C. hartmannianum (Schweinf)
C. procera (Aiton) Dryand
E. hirta L.
K. senegalensis (Desv) A. Juss
P. aculeata L.
P. glabrum Willd
R. communis L.
S. dubium Fresen
S. italica Mill.
S. persica L.
T. brownii Fresen
T. laxiflora Engl.
T. nilotica (Ehrenb.)Bunge
T. terrestri L.
V. amygdalina Delile
X. brasilicum Vell.
Also noteworthy the combrataceae family; C. hartmannianum (bark), T. brownii (wood and bark) and T. laxiflora demonstrated inhibitory activity against P. gingivalis with MIC values 2 mg/ml or less, except 50% ethanol extract of C. hartmannianum (wood) had no activity up to 4 mg/ml. This family has a wide range of tannins, flavonoids, terpenoids and stilbenoids [24, 25]. Flavonoids have been reported to be mainly active against Gram-negative bacteria . In this study, methanolic extract of C. hartmannianum (bark) exhibited good activity against P. gingivalis (MIC 0.5 mg/ml), and this was in agreement with Eldeen and Van  who reported that bark of C. hartmannianum inhibited the growth of Gram-negative bacteria at a concentration less than/or around 1.56 mg/ml.
The positive control (chlorhexidine) showed a significant inhibitory activity compared to the other extracts. However, chlorhexidine has several side effects such as undesirable tooth discoloration, unpleasant taste and causing dryness and burning sensation in the mouth, leading to patient dissatisfaction [28, 29].
Inhibitory activities of selected methanolic plants extracts against MMP-9
Several therapeutic strategies, based on targeting different pathways of the pathogenesis of periodontal disease, have been put forward. In this regard, a number of authors proposed that periodontitis progression could be hampered by successfully inhibiting both bacteria and host-derived proteinases involved in connective tissue destruction of the periodontium [30, 31].
From our previous study to explore a natural agent for preventing and treatment of dental cavity, seven Sudanese methanolic extracts namely; T. laxiflora (wood), Tamarix nilotica (stem) and bark of Khaya senegalensis‚ Acacia seyal var. fistula, Acacia seyal var. seyal, C. hartmannianum and T. brownii showed potent inhibitory activity against glucosyltransferase enzyme that promotes the binding of cariogenic bacteria on the teeth (Additional file 1: Table S1). Therefore these seven methanolic extracts were selected for assayed their ability to inhibit the MMP-9 enzyme.
Inhibitory activities of compounds isolated from C. hartmannianium bark against P. gingivalis and MMP-9
In the present study, some plants belong to combretaceae family revealed good inhibitory activities against P. gingivalis and MMP-9; such as methanolic extracts of T. laxiflora, C. hartmannianium (bark) and T. brownii (bark). The potency of T. laxiflora was probably due to the presence of terchebulin and flavogalonic acid dilactone in wood at high concentration . Kosei et al.  isolated gallic acid, punicalagin, terchebulin, ellagic acid 4-O-α-L-rhamnopyranoside, ellagic acid, and 3, 4, 3’-tri-O-methylellagic acid from methanolic extracts of T. brownii bark. However, there is no data was reported in literature regarding the isolated compounds from C. hartmannianium species bark, which makes it a potential candidate for further separation and isolation of compounds.
Among antimicrobial active compounds isolated from Combretum spp. are; combretastatins, acidic tetracyclic and pentacyclic triterpenes/triterpenoids, ellagitannins, phenanthrenes, flavonoids and saponins [37, 38]. C. hartmannianum gave good activity against Gram-positive and Gram-negative bacteria, and the most of the activity was found in water and methanol extracts. Additionally, the extracts of C. hartmannianum were found to be active against enzymes such as reverse transcriptase and tyrosine kinase .
Minimum inhibitory concentration (MIC) and matrix metalloproteinases −9 (MMP-9) inhibitory activities of isolated compounds from Combretum hartmannianium bark
*IC50 against MMP-9 (μM)
6.7 ± 1.5a
Flavogallonic acid dilacton
36.1 ± 7.5b
Terchebulin and flavogalonic acid dilactone had moderate antibacterial activity with MIC values of 500 and 1000 μg/ml, respectively. Previous studies showed that flavogalonic acid dilactone, terchebulin and punicalagin isolated from Terminalia spp. demonstrated antibacterial activity against P. acnes and Helicobacter pylori in a range between 125 to 250 μg/ml [15, 41]. Terchebulin demonstrated more potent activity (6.7 μM) than flavogalonic acid dilactone against MMP-9. Moreover, terchebulin has more reliable activity than chlorhexidine that inhibits MMP-9 at the IC50 25.2 μM . Furthermore, Arabaci et al.  found that chlorhexidine had a few genotoxic and cytotoxic effects on human lymphocytes. Studies of the in vitro cytotoxic activity on mouse fibroblasts of terchebulin and flavogalonic acid dilactone showed activity at minimum cytotoxic concentration of ≥1500 μg/ml (1348, 3192 μM respectively) . To the best of our knowledge, hydrolysable tannins mainly, terchebulin and flavogalonic acid dilactone were isolated from C. hartmannianium bark for the first time during this study.
Our study demonstrated that some methanolic crude extracts of Sudanese medicinal plants possessed good combined activities against P. gingivalis and MMP-9. Moreover, this study provided new information on terchebulin and flavogalonic acid dilactone which were isolated from methanolic extracts of C. hartmannianium bark, indicating that they possessed interesting inhibitory properties against P. gingivalis and MMP-9, and this may be useful for the prevention and treatment of periodontal diseases. Further studies are recommended to investigate the mechanisms of action of these isolated compounds, toxicity and their usefulness as a source of new components in mouthwashes and toothpastes.
The authors would like to thank prof. Abdelkhalig Muddathir and Dr. Farouk Hassan Eltahir for the English editing of the manuscript.
No fund was available.
Availability of data and materials
E.A.M.M participated in the design of the study, antibacterial, enzyme assay, isolation of compounds and write the manuscript. A.M participated in collection, extraction of plant samples and helped to draft the manuscript. T.M supervised and designs this study. All the authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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