Abstract: In the present study, 36 plant extracts, belonging to 6 families from Burkina Faso were used to evaluate their glutathione-S-transferase (GST), acetylcholinesterase (AChE), carboxylesterase (CES) and xanthine oxidase (XO) inhibitory activities and their phenolic, tannin and flavonoids contents by using spectrophotometrical methods. At 100 μg mL-1, Lippia chevalieri, Eclipta prostrata, Lantana camara and Indigofera pulchra extracts showed the best percentage of inhibition by regulating GST, AChE, CES and XO activities, respectively. The phytochemical investigations showed that all plant extracts were rich in biological compounds, namely phenolic, tannin and flavonoids. Particularly Cassia mimosoides extract presented the best phenolic, tannin and flavonoid contents. This result indicated that phenolic from Ceasalpiniaceae, flavonoids from Combretaceae and tannin from Verbenaceae contribute significantly to the inactivation of CES, AchE and GST, respectively. However, no significant correlation was found between polyphenolic compounds content and XO inhibitory activity. Present findings could partially justify the traditional uses of these plants in the treatment of mental disorders, gout, painful inflammations and cardiovascular diseases.
INTRODUCTION
Glutathione-s-transferase (GST), Acetylcholines-terase (AChE) and Carboxylesterase (CES) are known for their participation on the development of Alzheimer’s disease (AD), gout, cancer and cardiovascular diseases (Djeridane et al., 2008; Hayeshi et al., 2007). These diseases are becoming now-a-days a threat to public health (Orhan et al., 2004). Some previous investigations showed that the inhibition of these enzymes was a good way for biological molecules research against those diseases (Orhan et al., 2004; Trumbeckaite et al., 2006; Wu and Ng, 2008; Havlik et al., 2010). Furthermore, it is well demonstrated that medicinal plants were a promising way for obtaining biological molecules (Djeridane et al., 2006). In Burkina Faso, some ethnobotanical investigations showed the popular use of medicinal plants in the treatment of cardiovascular diseases, gout and cancer (Nacoulma, 1996). Table 1 shows the traditional uses of 36 well-known plants from Asteraceae, Caesalpiniaceae, Combretaceae, Fabaceae, Lamiaceae and Verbenaceae families.
Previous biological investigations showed that E. prostrata and L. camara extracts possess AChE inhibitory activities (Vinutha et al., 2007). Nevertheless, the phytochemical studies demonstrated that flavonoids, tannins, polyphenolic were founds in T. indica, Indigofera species, E. prostrata and O. americanum extracts (Paarakh, 2010; Lamien-Meda et al., 2008; Bakasso et al., 2008; Gopiesh and Kannabiran, 2007; Vieira et al., 2003). Other previous results have shown the secondary metabolite such as flavonoids and tannins presented some interesting enzyme inhibition activities (Senol et al., 2010).
| Table 1: | Traditional use, part used and herbarium numbers of plants |
| L: Leaves, SL: Stem leaves, WP: Whole plant | |
Flavonoids for example are known to inhibit a number of enzymes such as xanthine oxidase, acetylcholinesterase, glutathione-s-transferase and carboxylesterase (Bonesi et al., 2010; Iswantini et al., 2009; Senol et al., 2010; Soeksmanto et al., 2010). But, according to present knowledge, there is little scientific information about the enzyme inhibition properties of most of these plants. In the present study, 36 medicinal plant extracts, according to their traditional utilizations, were used to assess: (1) their AChE, GST, CES and XO inhibitory capacities and (2) their phenolic, tannin and flavonoid content.
MATERIALS AND METHODS
Chemicals: Reagents come from Sigma Aldrich Chemie GmbH, Germany: L-Glutathione reduced (GSH), glutathione-S-transferase (GST) from rate liver, 1-chloro-2,4-dinitrobenzene (CDNB), Albumin from bovine serum (BSA), potassium phosphate monobasic (KH2PO4) and dibasic (K2HPO4). Acethylcholinesterase (AChE) from electric eel, acethylcholine iodide (ATCI), 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), tannic acid, gallic acid, quercetin were provided from Sigma-Germany. HCl and sodium carbonate were from Labosi-France. Folin-Ciocalteu reagent was from Sigma-USA. Carboxylesterase from pig liver, Xanthine oxidase, DMSO and Tween were purchased from Sigma-Aldrich Chemie GmbH (Germany). Aluminum trichloride (AlCl3), Na2HPO4 and NaH2PO4 were purchased from Sigma-Aldrich Chemie (Steinheim, Germany).
Plant materials: Plant materials constituted of 36 medicinal plants from interior of Burkina Faso were collected at Ouagadougou in July 2006. The plants were botanically identified by Professor Millogo-Rasolodimby from Ecology Laboratory of the University of Ouagadougou. Voucher specimens (Table 1) were deposited in the herbarium of the Laboratory of Biology and Vegetal Ecology, UFR/SVT of the University of Ouagadougou.
Preparation of plant extracts: Tissue samples (leaves, stems-leaves and whole plant) of each plant were dried at room temperature and ground to fine powder; using a grinder. The extraction was processed using ten gram of each sample in 3x100 mL by technical methanol steeping during one night. The extracts were filtered and evaporated until they dry.
BIOLOGICAL ACTIVITY
Acetylcholinesterase activity: The AChE inhibition was conducted according to the protocol described by Lopez et al. (2002) with some modifications. Briefly described, the assay mixture consisted of 200 μL of Tris-HCl (50 mM pH8), 0.1% BSA buffer, 100 μL of extracts solution (final concentration: 100 μg mL-1) and 100 μL of AChE (0.22 U mL-1). The mixture was incubated at room temperature for 2 min before adding 500 μL of DTNB (3 mM) and 100 μL of substrate (ATCI 15 mM). The developing yellow color was measured at 405 nm after 4 min (Cecil CE 2041, England). Galanthamine was used as a positive control at a final concentration of 100 μg mL-1 in the assay mixture. AChE inhibitory activity was expressed as percent inhibition of AChE, calculated as:
where, A is the change in absorbance of the assay without the plant extract and B is the change in absorbance of the assay with the plant extract.
Inhibition of glutathione-S-transferase: GST inhibitory assays were conducted as Habdous et al. (2002).
Assay of xanthine oxidase activity: The XO inhibitory activities were measured spectrophotometrically by using Filha et al. (2006) procedure with some modifications. The extracts were directly dissolved in phosphate buffer-MeOH (1%) and screened for XO inhibitory activity at final concentration of 100 μg mL-1. The assay mixture consisted of 100 μL of extracts, 300 μL of phosphate buffer (0.2 M pH9) and 100 μL enzyme solution (0.28 U mL-1 in phosphate buffer). The mixture was incubated at room temperature for 2 min. Then, the reaction was initiated by adding 500 μL of xanthine solution (0.15 mM in phosphate buffer) and the change in absorbance was recorded at 295 nm for 2 min at room
temperature. Allopurinol was used as a positive control at a final concentration of 100 μg mL-1. The results were expressed as percent inhibition of xanthine oxidase, calculated as:
where, A is the change in absorbance of the assay without the plant extract and B is the change in absorbance of the assay with the plant extract.
Assay of carboxylesterase activity: The method of Djeridane et al. (2008) was used with some modifications. Test solution contained 400 μL of Tris-HCl (50 mM pH8) buffer, 100 μL of plant extract at final concentration of 100 μg mL-1. One hundred of enzyme solution (0.027 U mL-1) and 400 μL of 4-nitrophenyl (1 mM) was added after incubation at 3 min. The absorbance was read at 414 nm. Ascorbic acid (50 μg mL-1) was used as reference. The results were expressed as percent inhibition of CES, calculated as:
where, A is the change in absorbance of the assay without the plant extract and B is the change in absorbance of the assay with the plant extract.
Determination of polyphenolics compounds
| • | Determination of total phenolic content: The total phenolics of plant extract were determined by the Folin-Ciocalteu method (Lamien-Meda et al., 2008) |
| • | Determination of tannins content: Tannins content was determined according to the European Commission (2000) |
| • | Determination of flavonoids contents: The total flavonoids were estimated according to the Dowd method as adapted by Lamien-Meda et al. (2008) |
Statistical analysis: The data are expressed as the Means±Standard Deviation (SD) of three determinations. Statistical analysis (ANOVA with a statistical significance level set at p< 0.05 and linear regression) was carried out with XLSTAT 7.1.
RESULTS AND DISCUSSION
Biological investigations
AChE inhibitory activity: The highest inhibition activities were
obtained with L. chevalieri, C. mimosoides, C. nigricans and C. singueana
extracts. The lowest inhibitory effects were obtained with C. absus,
C. paniculatum, V. colorata and B. engleri extracts.
| Table 2: | Acetylcholinesterase (AChE), glutathione-S-transferase (GST), carboxylesterase (CES) and xanthine oxidase (XO) Percentage Inhibition Activities |
| ND: not determined; Result within each column with different letters (a-s) differs significantly (p< 0.05). | |
All extracts of AChE inhibitory activities were less than Galanthamine inhibitory effect. Previous studies showed E. prostrata and L. camara extracts AChE inhibitory activity (Vinutha et al., 2007). According to the cholinergic hypothesis memory impairment in patients suffering from Alzheimer’s disease is a result of decreased levels of the neurotransmitter acethylcholine (ACh) in the cortex. In the healthy brain AChE is the most important enzyme regulating the ACh level (Ahmad et al., 2003; Adsersen et al., 2007). In this way, it would be relevant to search for substance capable of inhibiting AChE activity in Alzheimer’s disease patients to increase their ACh levels such as Galanthamine that is used in this treatment. Table 2 showed the 36 extracts AChE inhibitory activities that were compared with Galanthamine activity. Present result could probably justify the plant traditional uses in cancer treatment. Among the five species, which inhibited AChE at more than 25%, four are included in Caesalpiniaceae family. In this way, Caesalpiniaceae is most indicated to search acetylcholinesterase inhibitors.
GST inhibitory activity: Table 2 showed 36 extracts GST inhibitory activities. The best activities were obtained with E. prostrata, L. chevalieri, L. rhodesiensis and V. colorata extracts. The lowest inhibition were obtained with T. indica, C. italica and C. macrocalyx extracts. Previous investigations showed that GST was implicated in tumor cell resistance to antitumoral drug treatment (Hayeshi et al., 2007).
| Table 3: | Result of polyphenolic quantification |
| TP: Total phenolic content TF: Total flavonoid content TN: Tannins content. Result within each column with different letters differs significantly (p<0.05) | |
| Table 4: | Correlative study between polyphenolics compounds and the enzymes inhibition |
So, the inhibition of GST activity become a promising way to develop antitumoral drugs, particularly, drugs from medicinal plant. Present results demonstrated that these extracts contained some compounds with GST inhibitory activities singularly in E. prostrata extract. These observations could be partially supported by the plant traditional use in cancer treatment indicated in Nacoulma (1996) ethnobotanical investigations. Between the six families which were studied Fabaceae species have not presented any inhibition for the enzymes.
CES inhibitory activity: The CES inhibitory effect of extracts were shown in Table 2. The best inhibition activity was found with C. mimosoides, C. crotonoides, C. singueana and L. camara extracts. Interestingly, ascorbic acid and L. camara extract have shown similar CES inhibition activities. Verbenaceae was the most CES inhibitor among the families. CES are enzymes omnipresent (high levels in a large array of animal tissues) responsible of the detoxication to numerous endogen and xenobiotic. Now-a-days, their biological role are not clearly delimited. For example CES also hydrolyse aspirin and some anti-cancerous such as chemotherapeutic agents (Djeridane et al., 2008). In this way, their inhibition can contribute to strengthen these drug effects (Crow et al., 2008; Rodinbo et al., 2003). Present results indicated that different extracts contain some molecules which were able to inhibit this enzyme, particularly in L. camara extract, while species from Lamiaceae and Fabaceae seem to be poor in CES compound inhibitor. Plant extract CES inhibitory properties evaluated in the first time could partially justify the traditional uses found in Burkina Faso (Nacoulma, 1996). In the CES inhibition, some extracts of plants show that they possess interesting activities as compared to ascorbic acid (56.72±0.85). L. camara (Verbenaceae) which has the best inhibition activity, inhibited CES at 56.20%.
XO inhibitory activities: The extracts XO inhibitory activities compared to allopurinol effect were shown in Table 2. Sixteen extracts inhibited XO at a level higher than 25%. The highest inhibitions were obtained with C. absus, I. macrocalyx and I. pulchra extracts (72.11±2.55, 72.82±1.45 and 77.44±0.73%, respectively) and the lowest are obtained with C. adenogonium, H. suaveolens, C. aculeatum. The Indigofera family presented a greater XO inhibitory effect than other families. But, all extracts have not reached the reference compound in XO inhibitory activity, i.e., allopurinol with 96.38±0.58%. The role of xanthine oxidase is to catalyze the oxidation of hypoxanthine to xanthine and generates uric acid, hydrogen peroxide and superoxide anion (Wu and Ng, 2008). Clinical reports have shown that uric acid is the key factor of risk of gout and cardiovascular disorders, nephrolithiasis and diabetes (Havlik et al., 2010; Iswantini et al., 2009; Gagliardi et al., 2009). Thus, xanthine oxidase inhibition is useful in the prevention and/or treatment of hepatic diseases and gout, and also for the reduction of harmful hydrogen peroxide and superoxide anion productions. Present results suggest that respective extracts contained XO inhibitors compounds. These observations could justify the traditional use of particularly I. pulchra (cellulites), C. crotonoides and D. tomentosa (rheumatism). No species of Verbenaceae family inhibited XO but all species of Fabaceae family inhibited at more than 30%.
Phytochemical analysis: The total tannin and phenolic content of the extracts were determined spectrophotometrically (Table 3), because polyphenols present in vegetable and fruit are responsible for many biological activities (Arct and Pytkowska, 2008; Soobrattee et al., 2005). Total phenolics of extracts were ranging from 51.3±0.49 to 10.03 ± 0.75 mg GAE/100 mg. The highest values of total phenolics were in the following order: C. mimosoides> C. micranthum = C. singueana. The lowest ones were found in L. camara and C. americanum with 10.03±0.75 to 10.15±0.37 mg GAE/100 mg extract plant, respectively. Among the 36 extracts of plants analyzed, C. mimosoides (33.60±0.57 mg TAE/100 mg extract) and C. nigricans (20.73±0.53 mg TAE/100 mg extract) showed the highest tannin contents. The lowest values were found in L. camara and C. occidentalis extracts. Previously, the polyphenolic contents were evaluated in Fabaceae family extracts (Bakasso et al., 2008), T. indica extracts (Lamien-Meda et al., 2008) and the tannin content in E. prostrata extract (Gopiesh and Kannabiran, 2007).
Many flavonoids possess anti-tumor activity against various human cancer cell lines and xenograft systems of human tumors, suggesting that they may be promising anticancer agents (Zeng et al., 2009; Paarakh, 2010; Soeksmanto et al., 2010). In this way, flavonoids contents were estimate in 36 plant extracts (Table 3). Total flavonoid contents varied from 30.43±0.28 to 0.29±0.01 mg QE/100 mg plant extract. The best flavonoids contents were obtained with I. pulchra, E. prostrata, C. mimosoides extracts and the lowest contents with A. hispidum, L. martinicensis, V. colorata, T. procumbens and extracts. In previous phytochemical investigations, Bakasso et al. (2008), Lamien-Meda et al. (2008), Gopiesh and Kannabiran (2007) and Vieira et al. (2003) have found the flavonoids in Indigofera species, T. indica, O. americanum and E. prostrata extracts, respectively.
Relationship between enzyme activities, total phenolic, tannin and flavonoids contents: In the present study we were interested by the evaluation of the phenolic, flavonoid and tannin of extracts capacities to inhibit the different enzymes (Table 4). Tannin from Fabaceae, Caesalpiniaceae, Verbenaceae and Asteraceae families extracts contributed significantly to inhibit AChE, CES and GST, respectively. The same observation was notified concerning the flavonoids from Lamiaceae, Combretaceae, Asteraceae and Verbenaceae on these enzyme inhibition activities. Only the phenolic from Caesalpiniaceae and Verbenaceae families extracts presented a significant correlation to CES inhibitory activities. Nevertheless any correlation was found concerning the XO activity and the metabolites from different family extracts, although this enzyme was inhibited, interestingly, by indogofera species (Fabaceae) extracts. These results indicated the enzyme inhibition did not only depend on the metabolite quantities. Consequently, L. camara extract presented little phenolic as tannin contents (10.03 mg GAE/100 mg and 0.48 mgTA/100mg) with interesting AChE, GST and CES inhibitory activities. Also C. absus extract was a good XO inhibitor with little phenolic and flavonoids contents.
These are several reasons to explain the ambiguous relationship between the inhibitory potency and the phenolic, tannins and flavonoids. The total phenolics content did not include all the possible inhibitors; the synergism among the inhibitors in the mixture accounted for the inhibition but was not only dependent of the concentration of individual inhibitors but also on the structure and interaction among them. Previous studies showed the structure relationship of flavonoids in CES (Stocker et al., 2004), GST (Van Zanden et al., 2004), AChE (Ji and Zhang, 2006) and XO (Nagao et al., 1999) inhibitory activities. On the other hand, the method used to quantify the flavonoids was limited to flavone and flavonol (Meda et al., 2004). A detailed examination of phenolic of different plant extracts is necessary for a comprehensive assessment of the individual compounds enzyme inhibitory ability.
CONCLUSION
This study has showed that the methanolic extracts of 28 on 36 species are potential inhibitors of AChE, GST, CES and XO which could support the traditional uses of these plants in the treatment of mental disorders, gout, painful inflammations and cardiovascular diseases. Fabaceae family is particularly important as a source of natural XO inhibitors. Considering the correlation analysis, Combretaceae and Caesalpinaceae could also be relevant sources of inhibitors. Some plant extracts were effective inhibitors for three of the four enzymes at 25% such as E. prostrata, L. chevalieri, C. nigricans and C. mimosoides. Those activities seem to be partially correlated to the flavonoid and tannin contents. Those plant species were indicated for new molecules to relieve the diseases, which involved these enzymes. Future studies aim to isolate and identify these active constituents that exhibit significant AChE, GST, CES and XO inhibitory activity through bioassay-guided fractionation.
ACKNOWLEDGMENTS
This project was supported by the International Fondation of Sciences through the grant F3979-1 allowed to Dr. Martin Kiendrebeogo. The authors gratefully acknowledge the financial support by the Conseil Interuniversitaire de la Communauté française de Belgique/ Commission Universitaire pour le Développement (CIUF/CUD).