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Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by an alkaloid extract from Combretum molle
© Moyo et al.; licensee BioMed Central Ltd. 2014
Received: 6 February 2014
Accepted: 30 June 2014
Published: 5 July 2014
Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated.
H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates.
A specific activity of 24 μmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 μg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 μg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 μg/ml and Ki ′ = 9.2 μg/ml.
The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.
The inflammatory process may be defined as a sequence of events that occurs in response to noxious stimuli, an infection or trauma. It is the first response of the immune system to infection or irritation and is a protective attempt by the organism to remove injurious stimuli and initiate the healing process. Inflammation is a self-defense reaction in its first phase and, hence, is regarded as the main therapeutic target and the best choice to treat the disease and alleviate symptoms. Inflammation plays an important role in various diseases, such as rheumatoid arthritis, atherosclerosis and asthma, which all show a high prevalence globally.
The inflammatory process is mediated by prostaglandins which are synthesized from arachidonic acid. Prostaglandin D2 is an acidic lipid mediator which is responsible for the regulation of body temperature, hormone release, olfactory reaction, sleep, prevention of platelet aggregation and pain responses. PGD2 interacts with two types of G protein coupled receptors that is DP1 and DP2. The DP2 receptors are also known as chemo-attractant receptor homologous on T helper 2 cells (CRTH2). DP1 receptors are found on murine and dendritic cells and DP2 on Th2, eosinophils and basophils. DP2 receptors mediate eosinophil chemotaxis and are involved in Th2 related inflammation.
PGD2 is formed by the action of two types of prostaglandin D2 synthase isoforms that is lipocalin and hematopoietic type. Lipocalin prostaglandin D2 synthase is found in the central nervous system, testis and human heart. Production of PGD2 is mainly mediated by a glutathione- dependent hematopoietic prostaglandin D2 synthase (H-PGDS). Hematopoietic prostaglandin D2 synthase (H-PGDS) is widely distributed in antigen-presenting cells, T helper (Th2) lymphocytes, mast cells, and megakaryocytes, where it selectively metabolizes cyclooxygenase-derived PGH2 to PGD2. H-PGDS is characterised as a member of the Sigma class of GST gene family which catalyses the conjugation of glutathione (GSH) to an electrophilic substrate. The high specificity of the enzyme for the production of PGD2 is attributed to the unique structure of the catalytic unit which is deep and wide unlike the catalytic units for other GSTs which are narrow and shallow. Inhibition of H-PGDS has been shown to be very protective in mouse models with allergic airway inflammation. Thus, by analogy H-PGDS appears to be a promising target for the design of anti-allergic and anti-inflammatory drugs.
Non - steroidal anti - inflammatory drugs (NSAIDs) are among the most commonly used drugs worldwide to aid in treating inflammatory conditions. It is estimated that up to 60% of individuals taking NSAIDs will experience side effects and also some of the NSAIDs such as naproxen have been shown to contribute to a 50% higher risk of heart attack and stroke with long term use. Due to these side effects alternative forms of medicine may help in managing inflammatory conditions.
Ethnobotanical knowledge on plants possessing anti-inflammatory and analgesic properties can open up to new drugs in inflammatory disorders. Medicinal plants constitute an effective source of medicines and herbal medicines have been shown to have profound utility with about 80% of rural population depending on it for their primary health care. In Zimbabwe the leaves of Rhus dentate, Ochna pulchra have been used to treat stomach pains. Chimbwidi and Dalbergia melsnoxylon have also been used to treat asthma.
Ojewole, found analgesic, anti-inflammatory and cardiovascular effects of mollic acid glucoside isolated from C. molle leaves and antiprotozoal activity from the acetone extract of leaves from the same plant. C. molle was found to have anti-asthmatic and anti-tussive activities. In an investigation of the biological activity of different Combretum species, C. molle was found to have both anti-inflammatory and anti-schistosomal activity. Inflammatory diseases are a major and worldwide problem. An important mediator of inflammation is PGD2 which is produced from PGH2 by H-PGDS. Very few studies have been done on H-PGDS, which is an enzyme that has been linked to the inflammation process. Of the few studies done it was shown that H-PGDS is associated with inflammation and allergic reactions[5, 9].
According to other studies on Combretum species, alkaloids have been shown to have anti-inflammatory properties. The effects of un-fractionated alkaloids from C. molle were, thus, determined in this study. The main objective of this study was to investigate the effects of alkaloids isolated from C. molle on H-PGDS.
Human recombinant H-PGDS was a kind gift from Professor Bengt Mannervik (Uppsala, Sweden). Ethacrynic acid, cibacron blue, CDNB, GSH were products of Sigma Aldrich. All chemicals unless stated otherwise were purchased from Sigma-Aldrich (Steinheim, Germany).
Plant collection and preparation
The leaves of C. molle were collected from Centenary in Mashonaland Central Province of Zimbabwe. The plant was authenticated and classified by Mr. Christopher Chapano, a taxonomist at the National Herbarium and Botanic Gardens (Harare, Zimbabwe). Herbarium plant samples were kept at the Department of Biochemistry, University of Zimbabwe, Harare, Zimbabwe. Leaves of Combretum molle were separated from the plant and then dried at an ambient temperature of 50°C in an oven (Memmert, SRG, SchwaBach, , Germany). The dried leaves were ground to a powder using a two speed blender (BL2, ABB, Moulinex, France) so as to optimize the solvent contact during the extraction process. The powders were weighed on a digital balance (Kern EG, Balingen, Germany) and their masses were recorded.
The leaf powder was extracted with ethanol and acetone. An aliquot of 5 g of the powdered sample was weighed on a balance (Kern and Sohn Co., Balingen, Germany) and mixed with 25 ml ammonia and 50 ml 10% ethanol. The mixture was mixed thoroughly on Vortex mixer (Thermolyne Maxi Mix II, IOWA, USA) and placed in a water bath at 40°C for 10 minutes. The mixture was then filtered through a Whatman filter paper 1 and air dried under a fan. The powder obtained was packed in 50 ml test tubes and stored at 25°C for future use.
Expression and purification of H-PGDS
H-PGDS was expressed from a pJexpress 401plasmid in E.coli XL1-blue cells. The gene also coded for hexahistidine tail. Luria Bertani (LB) medium was prepared and kanamycin was added to a final concentration of 50 μg/ml. A volume of 5 ml of the incubated H-PGDS containing E. coli cells was added to each of 2 flasks each containing 500 ml media. The expression of H-PGDS was induced by the addition of isopropyl-beta-thiogalactopyranoside (IPTG) after the absorbance (OD) of 0.4, at λ = 600nm was reached and IPTG was added to make a final concentration of 0.2 mM. The cells were then incubated at 160 rpm at 37°C for a further 15 hours in a SI 300 Lab companion, (Jeio Tech, Seoul, Korea). A pellet was obtained after centrifugation at 3 000 rpm for 5 minutes using a Hettich Rotofix 32 A centrifuge (Tuttlingen, Germany). The pellet obtained from centrifuging was lysed with a lysis buffer (pH 8 phosphate buffer 50 mM, 0.3 M sodium chloride, 10 mM imidazole, 1 mg/ml lysozyme).
This mixture was sonicated using a sonicator (Vibra cell, New York, USA) 2 × 20 s treatment stopping at 2 minutes interval to avoid damaging the protein by heating. Phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 170 μM to inhibit proteases. This mixture was then centrifuged using a Beckman Optima LE-80 K ultracentrifuge, (Beckman instruments, California, USA) at 105 000 × g for 1 hour. The supernatant was retained while the pellet was discarded. Protein was then purified by nickel immobilized metal affinity chromatography using Ni Cam affinity resin following the manufacturer’s instructions Sigma-Aldrich (Steinheim, Germany). The fractions collected from the column were tested for H-PGDS activity using CDNB as a substrate. The fractions that exhibited activity were pooled and concentrated using an IVSS Vivapore 10 /20 concentrator (VP2001 Satorius Stedim Biotech, Stonehouse, UK) with a molecular weight cut off of 7500 daltons. The concentrated solution was then dialyzed against 2 × 5 L of dialysis buffer (50 mM sodium phosphate pH 8, 1 mM EDTA, 0.2 mM DTT, 0.02% NaN3) using a membrane with a molecular cut off of 12 000 daltons.
The purity of the enzyme purification fractions was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), carried out on 15% slab gels using a Hoeffer SE Mighty Small II electrophoresis system (Hoeffer Scientific Instruments, California, USA). Protein bands were stained with Coomasie Blue-G.
Screening for Inhibition of H-PGDS by alkaloids from C. molle
The effect of C. molle alkaloid extract on H-PGDS was tested at 300 μg/ml. The enzyme activity was determined through the measurement of the conjugation activity with CDNB at 340 nm using SQ Single Beam Scanning UV/Visible Spectrophotometer (United Products and instruments Inc., USA) and was done in quadruplicate. For the determination of IC50, a 2 fold serial dilution of C. molle extracts were carried out from 0 to 300 μg/ml in a 96 well plate. The assay with CDNB was adapted for measurement of absorbance with a SpectraMax Plus microplate spectrophotometer equipped with a kinetics mode (Molecular Devices, Sunnyvale, California, USA) at 380 nm using an extinction coefficient of 7.825 μM-1 cm-1.
Determination of time-dependent effects
The incubation mixtures contained H-PGDS (final concentration 0.0625 μM), 0.2 M potassium phosphate buffer pH 7.4 with 0.2 mM EDTA, and (15, 30, 60 μg/ml) C. molle alkaloid extract. The incubations were carried out at a temperature of 30°C. The experiment was carried out at timed intervals from 0–1 hour, beginning with incubation for the 1 hour sample and lastly with the 0 hour time sample. Immediately, 20 μl of each sample was withdrawn and assayed for GST activity. These incubations were run in parallel with positive and negative controls. The negative control contained H-PGDS and buffer. Cibacron blue was used as the positive control.
Determination of kinetic constants for H-PGDS using CDNB and GSH as substrates
The effects of alkaloids from C. molle on the kinetics of H-PGDS were determined as described previously. Activity with CDNB was measured by determining the absorbance with a SpectraMax Plus microplate spectrophotometer equipped with a kinetics mode (Molecular Devices, Sunnyvale, CA, USA). The K m(app) and V max(app) were determined using GraphPad Prism™ version 5.00. The K i values with respect to GSH and CDNB, as well as the type of inhibition were determined. The type of inhibition was deduced by determination of trends of K m and V max values with increase in natural product concentration. To determine the trend, the means of the K m (or V max ) values with increase in inhibitor concentration were compared by performing a one-way ANOVA with Dunnett’s post test using Graph Pad 5.00 (Graph Pad Prism Inc. San Diego, CA, USA). The inhibition constant, K i , was determined by means of re-plots. The type of re-plot depends on the type of inhibition, for example, plotting 1/V max versus inhibitor concentration for non-competitive inhibition will give K i as the intercept on the baseline.
Data analyses were performed using GraphPad Instat software® (GraphPad Prism Inc. San Diego, CA, USA). Levels of significance were determined using ANOVA using the Dunnett post test were all columns of treatments were compared to the control. All data were expressed as mean ± standard deviation. P ≤ 0.05 values or less were considered to indicate statistically significant difference).
Coomassie blue staining and Western blot for poly-His tag
H-PGDS was expressed in E. coli and was purified by Nickel affinity chromatography. The H-PGDS was purified to homogeneity and a single band was obtained on SDS-PAGE analyses. The molecular weight of the protein was 23.4 kDa and the specific activity was 24 units/mg.
The effects of C. molle alkaloids on H-PGDS activity
Time-dependent effects of alkaloids on H-PGDS
The effects of the alkaloids on H-PGDS kinetics
The effects of alkaloids from C. molle on the kinetic properties of GST Sigma
171.2 ± 1.7
2366.8 ± 38.6
5.2 ± 0.04
2.25 ± 0.04
33.17 ± 0.06
1051.2 ± 1.03
162 ± 0.3
2281.3 ± 3.5
4.82 ± 0.12
2.00 ± 0.02
33.62 ± 0,80
1136.14 ± 6.70
149.5 ± 2.6
2218.6 ± 37.3
3.41 ± 0.007*
2.17 ± 0.007
43.82 ± 0.86
1020.19 ± 13.83
127.1 ± 5.3*
2118.4 ± 31.2
1.40 ± 0.02**
2.06 ± 0.02**
90.92 ± 4.81
1028.83 ± 5.96
115.6 ± 1.9***
2074.5 ± 3.2*
1.14 ± 0.06**
2.02 ± 0.02**
101.9 ± 3.94
1029.36 ± 10.33
Inflammation is a major worldwide health burden in both developing and developed countries. Chronic inflammation can lead to various conditions such as cancer, asthma, rheumatoid arthritis, atherosclerosis, periodontitis, and hay fever. The search for safe and efficacious agents for use in inflammatory conditions is ongoing with the objective to find new drugs which are efficacious but with few side effects. Conventional medicines have been shown to possess many side effects and, thus, effort is being put into research or new drugs. An important prostaglandin involved in inflammation is PGD2 which is produced by H-PGDS. H-PGDS negative mice generated by standard gene targeting technology showed diminished symptoms of disease indicating diminished inflammatory reaction in the absence of H-PGDS.
A considerable number of studies have suggested that extracts or active principles obtained from Combretum species have a broad spectrum of biological activities, including antibacterial, antiprotozoal, anticancer, cytotoxic, analgesic, anti-inflammatory, hepatoprotective and antiviral activities. C. molle is used in traditional medical practices in Zimbabwe to treat pain and inflammation. The aim of the present study was to collect information on the possible pharmacological and molecular basis for the efficacy of the plant alkaloids on the effective management of inflammation. The preliminary studies in our research group showed that the crude plant leaf extract of C. molle brought about 87% inhibition of the H-PGDS. An alkaloid extract from C. molle was then assayed for its activity against H-PGDS.
Alkaloids are abundant in the leaves of C. molle and have been reported to have significant pharmacological activities. Previous studies on this plant led to the isolation of triterpenoids glycosides, tannins, alkaloids, saponins, stilbenes, triterpene saponin oleanone trypetitepene, arjunolic acid and mollic acid glucosides which demonstrated cytotoxic, antifungal, antimicrobial and anti-inflammatory activity. According to the histochemical studies done in a previous study, the main constituents of C. molle leaves were found to be phenolics, flavonoids and alkaloids.
Analgesic and anti-inflammatory properties of mollic acid glucoside (MAG), an alkaloid, a 1α-hydroxycycloartenoid extracted from Combretum molle leaves have been investigated in mice and rats. The results of the laboratory animal study indicate that MAG possesses analgesic and anti-inflammatory effects in the mammalian models used. The author suggested that MAG possesses both centrally- and peripherally-mediated analgesic effects. In an investigation of the biological activity of different Combretum spp, C. molle was found to have both anti-inflammatory and antischistosomal activity. These findings may explain the traditional use of the plant against malaria and pain. This study also contributes to the validation of the popular use of this plant species in the treatment of inflammation.
H-PGDS is specific for and selectively and effectively isomerizes PGH2 to PGD2, thus, efforts are being put into searching for potential HPGDS inhibitors. The activities of other GSTs have been reported to be inhibited by S-hexyl glutathione (GSH) and its conjugation with 1-chloro- 2, 4-dinitrobenzene (CDNB). In the present investigation, C. molle alkaloids were tested for GST inhibition in vitro. The effect of C. molle alkaloids on H-PGDS was tested at 300 μg/ml concentration of the plant extract. The inhibition profile was concentration-dependent (Figure 4). Other data also showed concentration-dependent inhibition of cytosolic GSTs when Mitragyna speciosa extract was added into the reaction mixture. In that study, the methanolic extract showed the highest GSTs specific activity inhibition (61%), followed by aqueous (50%) and total alkaloid extract (43%), respectively. In this study, C. molle alkaloids reduced the enzyme activity by 70%, thus, showing that the fractions were potent inhibitors of H-PGDS. Compounds that inhibit GSTs can prove to be potent drugs. Since the whole leaf extract from C. molle was shown to be a potent inhibitor for H-PGDS, further studies to determine the IC50 values for alkaloids were carried out. C. molle alkaloid extract exhibited inhibitory effects on H-PGDS with an IC50 of 13.7 μg/ml. Since the IC50 value for the whole leaf extract was found to be 16.7 μg/ml, it suggests that the inhibitory effects in the leaf extract were mainly due to the presence of alkaloids in C. molle.
However, the IC50 value for the alkaloids was high as compared to other GST inhibitors namely, hematin (3.16 μg/ml), tributyltin bromide (2.2 μg/ml) and for S-hexylglutathione (7.8 μg/ml). The difference can be ascribed to the fact that this was a mixture and not pure compounds. In a previous study, it was found that the pure plant natural products ellagic acid and curcumin were potent inhibitors of GSTs with IC50 values of 0.6 and 0.9 μg/ml respectively and, therefore, purified phytochemicals maybe be more effective than mixtures.
To determine if alkaloids from C. molle had other modes of inhibition of H-PGDS, time-dependent incubations of the alkaloids with the enzyme were carried out. C. molle alkaloids failed to inactivate H-PGDS (Figure 4). Activity at the beginning of the reaction was concentration-dependent and lower than that of cibacron blue at all concentrations. Thereafter, with progression in time, the activity remained constant showing that the effects of the alkaloids were not time-dependent but were reversible.
It was shown that the plant extract exhibited an uncompetitive type of inhibition with regards to GSH as both Km and Vmax were decreasing and produced a Ki of 41 μg/ml. Thus, the extract binds to the enzyme – substrate complex only. The inhibition was non-competitive with respect to CDNB characterised by Ki value of 7.67 μg/ml and Ki ′ of 9.18 μg/ml. This suggests that the extract binds to the free enzyme and to the enzyme – substrate complex. In non-competitive inhibition, substrate can still bind to the enzyme-inhibitor complex. However, the enzyme-inhibitor-substrate complex does not proceed to form product and the value of Vmax decreases to a new value while the value of Km is unchanged. In a previous study, the Ki values for GSTs were 84.132 and 180 μg/ml respectively for T. diversifolia, C. rotundus and H. suavolens extracts and, hence, C. molle alkaloid extract with lower kinetic constants was a more potent GST inhibitor.
Although several studies have investigated the role of PGD2 in inflammation, the role of PGD2 in the host immune response has been scantly studied. Inflammation in H-PGDS knockout mice was found to be more severe during the onset phase arising from a cytokine imbalance which resulted in enhanced polymorphonuclear leukocyte and monocyte trafficking. Prevention of H-PGDS activity specifically either through gene knockout leads to impaired clearance of lymphocytes and macrophages from sites of inflammation. This may be the case when H-PGDS is inhibited by C. molle alkaloid extract. H-PGDS contributes to the production of the D and J series of prostanoids in the immune system and is involved in allergic inflammatory response. Since H-PGDS is present in mast cells, Th2 cells, and other leukocytes, it is thought to be mainly responsible for PGD2 production during allergic responses. Inhibition of H-PGDS will reduce the production of PGD2 and, hence, result in a decrease in allergies.
The substrate used in this study was CDNB which is not the physiological substrate for H-PGDS. However, C. molle alkaloid extract is more likely to possess anti-H-PGDS activity even in the presence of PGH2 the physiological substrate. The Km of H-PGDS obtained for PGH2 was 0.2 mM and in this study we obtained a Km of 2.25 mM for CDNB. Although these values are within a magnitude of difference, C. molle alkaloid extract is still likely to have some inhibitory effects on H-PGDS in the presence of PGH2. Further experiments using PGH2 as a substrate are needed to verify this claim.
In conclusion, alkaloids from C. molle were shown to have inhibitory effects on H-PGDS. The inhibitory effects were lower as compared to cibacron blue, a standard H-PGDS inhibitor. The alkaloids exhibited non-competitive and un-competitive inhibition of H-PGDS with respect to CDNB and GSH respectively. However, potent activity in vitro cannot be directly correlated to potent activity in vivo due to other factors such as metabolism within cells and the presence of other compounds in the cell which may prevent binding of the required compound to its target site. The effect of C. molle alkaloid extract on H-PGDS using PGH2 needs to be determined. This study has, therefore, identified alkaloids from Combretum molle with potential anti-inflammatory activity and may, therefore, serve as sources of lead compounds for anti-inflammatory drug development. The inhibitory effect of alkaloids from C. molle also validates the use of its extracts in traditional medicines to reduce inflammation.
This study was sponsored by the International Foundation in Sciences (IFS), Stockholm, Sweden; Grant Number F/3413-03F. Support from the International Science Programs (ISP) through the International Program in the Chemical Sciences (IPICS: ZIM01), Uppsala University, Uppsala, Sweden and the University of Zimbabwe Research Board (Harare, Zimbabwe) is also acknowledged.
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