NITRIC OXIDE INHIBITORY ACTIVITY OF STRYCHNOS SPINOSA (LOGANIACEAE) LEAF EXTRACTS AND FRACTIONS.

BACKGROUND: The study was aimed at determining the anti-inflammatory activity of fractions and extracts obtained from Strychnos spinosa leaves on a mediator of inflammation nitric oxide (NO).
MATERIALS AND METHODS: Leaves were extracted with acetone and separated into fractions with different polarities by solvent-solvent fractionation. The Griess assay was used to determine the nitric oxide (NO) inhibitory activity. Cellular toxicity was determined by "using the MTT reduction assay".
RESULTS: With the exception of the ethyl acetate fraction which had an IC50 >750 μg/mL, all extracts and fractions had significant nitric oxide-inhibitory activity. The most active being the water fraction, chloroform fraction and the dichloromethane/methanol extracts with IC50 values of 88.43 μg/mL, 96.72 μg/mL and 115.62 μg/mL, respectively. The extracts and fractions had low cytotoxicity on macrophage U937 cell lines.
CONCLUSION: Extracts and fractions of Strychnos spinosa leaves may be promising sources of natural anti-inflammatory agents. Findings obtained from this study showed that Strychnos spinosa leaves possess promising anti-inflammatory action and could be used in the treatment of inflammation-related conditions.

Introduction

Strychnos spinosa (Loganiaceae) has several medicinal properties including antimicrobial (Kubmarawa et al, 2007; Nwozo et al, 2007; Ugoh and Bejide, 2013; Isa et al, 2014a), antiplasmodial (Bero et al, 2009), antitrypanasomal (Hoet et al, 2007) and anthelmintic activities (Waterman et al, 2010). Previous studies had documented the anti-inflammatory activities of S. spinosa (Nhukarume et al, 2010; Isa et al, 2014b).

Nitric oxide (NO) serves as a signalling agent among tissues. NO also acts as a vascular relaxing agent, neurotransmitter and could inhibit platelet aggregation in mammalian tissues (Moncada et al, 1991; Lincoln et al, 1997). In addition to these physiological roles, NO is also generated during inflammatory and innunological responses (Moncada et al, 1991; Lincoln et al, 1997). The function of the molecule may be more complex (Moncada et al, 1991; Lincoln et al, 1997). It may be involved in innate immunity, in combating infectious organisms but it can also regulate specific immunity by inducing or regulating host immune cells functions and death. During some inflammatory conditions, NO is produced at very high levels in certain types of inflammation, and may induce toxic reactions against other animal tissues. It has been implicated as a pro-inflammatory agent in asthma (Coleman et al, 2001). Due to its inhibitory or apoptotic effects on cells it may also be an anti-inflammatory or immunosuppressive agent. The alleviative inflammatory symptoms, especially pain non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are the mainstream treatments. In some cases, these drugs have serious side effects. Natural products may contain potential immunomodulatory agents with fewer side effects.

In this study we determined the nitric oxide inhibitory activity and cytotoxicity of the leaf extracts of Strychnos spinosa a plant used traditionally to treat pain.

Materials and Methods

Plant material

Collection and identification of plant material

Fresh Strychnos spinosa leaves were collected from Sakara village located along new Jos road, Zaria. Samples of the fresh whole plant were sent to the Herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria for identification by a taxonomist (Musa Muhammad). The sample was stored with a voucher specimen number 900161. which compares with the Herbarium sample.

Preparation of extracts from S. spinosa leaves

Leaves were separated from stems, packed in an open mesh well-perforated bag and air-dried under shade at room temperature for 2 weeks. The air-dried leaves were ground into powdered form and stored in an air tight polyethylene bag in the dark until required.

Acetone, methanol and dichloromethane/methanol extracts

The extraction protocol has previously been described (Isa et al, 2014 a,b). Two kilogrammes of the dried ground powder of the leaves of the plant were extracted using six liters of acetone. The process was repeated three times and the residue was further extracted in six liters of methanol to yield the methanol extract. A separate 1 kg of leaf powder was extracted in three liters of dichloromethane/methanol (1/1, v/v) to provide the dichloromethane/methanol extract.

Fractionation of the acetone extract

The acetone was removed from the extract and 70 g was dissolved in a mixture of 700 mL of chloroform and 700 mL of water to obtain the water and chloroform fractions. The water fraction was further partitioned with 600 mL of n-butanol to afford n-butanol and water (Wat1) fractions. The chloroform component was concentrated to give solid extract in vacuo. Furthermore, the extract was dissolved in 100 mL of 10% water in methanol and partitioned with 2.5 L of n-hexane to obtain the hexane fraction and the residue of 10% water in methanol. The hexane fraction was dried completely and the residue of 10 % water in methanol was further diluted using distilled water (162.5 mL) to give 35% water in methanol. The 35% water in methanol was partitioned with 2 L of chloroform which yielded the chloroform and 35% water (wat2) fractions. Based on the thin layer chromatograms using the same eluent as Kotze and Eloff (2002), water (Wat1) and 35% water in methanol (Wat2) did not differ much and were combined into one fraction.

Alkaloids extraction

One kilogram of S. spinosa leaves was extracted with 600 ml of EtOAc-EtOH-NH4OH (96:3:1, v:v) and then extracted with EtOAc. The EtOAc was dried in a rotary evaporator and EtOAc was used to dissolve the extract. Thereafter, the mixture was mixed with 4% acetic acid to yield the EtOAc fraction. The acidic aqueous solution (pH 3-4) was basified to pH (8-9) with Na2CO3 and extracted three times with dichloromethane to give the crude alkaloids extract after removal of the solvent in vacuum.

Chemicals

The chemicals used were obtained from the following suppliers: Indomethacin and MTT (Sigma), quercetin, RPMI 1640 bottle (Sigma/Lonza/Highveld Biological), Penicillin/Streptomycin/Fongizone (PSF) (Highveld Biological), BS (Highveld Biological), U937 Cells (ATCC® CRL1593.2 ™), Griess reagent for nitrite (Lot BCBJ6549) Sigma, Germany; Nitric oxide (Lot S38919-456) Sigma, USA; lipopolysaccharide (LPS) from Escherica coli (L6529-1MG) Sigma, Germany;

Nitric oxide Assay

Roswell Park Memorial Institute Medium (RPMI) 1640 medium which was supplemented with 10% heated fetal bovine serum, containing 200 mM glutamine, 5.5 ml Penicillin-Streptomycin Amphotericin B solution, 10,000 units/ml Penicillin G Sodium Salt, 10 mg/mL Streptomycin Sulphate and 25 μg/mL Amphotericin B) were used to culture human macrophage U937 cell line. Cells were maintained in a 5% CO2 atmosphere with 95% humidity where cultured medium was changed three times a week. A sample of the culture was centrifuged, fresh medium added centrifuged again and the cell pellet, re-suspended in 10 mL growth medium to make a single cell suspension. Cells were counted and diluted with medium to yield a final concentration of 1 × 105 U937 cells/ml. Of this suspension a volume of 100 μl containing 1 x 105 cells was seeded in each 96-well microplate and allowed to adhere for 1 hr at 37°C in 5% CO2. The medium was then replaced with fresh medium containing 5 μg/ ml of LPS together with test sample initially dissolved in DMSO and then diluted with the growth medium to different concentrations before incubation for 48 hrs. NO production was measured by determining the accumulation of nitrite in the culture supernatant using the Griess reagent (Kim et al, 1995). Fifty microliters of each supernatant was mixed with the same volume of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrocholide in water); absorbance of the mixture at 550 nm was determined with a SpectraMax 190, Molecular devices microplate reader.. Standard calibration graphs were prepared using sodium nitrite as standard. Nitrite levels in samples were read off from the standard sodium nitrite graph.

Cytotoxicity Assay

The 3-(4,5-dimethyl-2- thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) colorimetric method was used to determined cytotoxicity (Mossman, 1983). Incubating test samples after 48 hrs with test samples, cells were washed with 150 μL phosphate-buffered saline (PBS) and fresh MEM (200 μL) was added to each well. After incubating microplates at 37°C for a further 4 hr, the medium in each well was carefully aspirated, without disturbing the MTT formazan crystals in the wells. The crystals were dissolved by adding 50 μL DMSO to each well and the plates gently shaken until the MTT solution was dissolved. The amount of MTT reduction was measured immediately by detecting absorbance in a microplate reader (Versamax, Molecular Devices) at a wavelength of 570 nm, and a reference wavelength of 630 nm. The wells in column 1, containing medium and MTT but no cells were used to blank the plate reader. The LC50 values were calculated as the concentration of test sample resulting in a 50%-reduction of absorbancompared to untreated cells.

Statistical analysis

All experiment were conducted in triplicate and the results are expressed as means ± S.D. Analysis of variance was used to show statistical significance followed by Dunnett’s post-hoc test. P-values of less than 0.05 were considered statistically significant. The tests were performed using Statistical Package for the Social Sciences /version 20.

Results

The extracts prevented the production of NO dose-dependently (Figures 1 and 2). The activity of NO as shown by the IC50 values is depicted in Table 1 and the selectivity index in table 2. The extracts had IC50 values between 88.4-325.1 μg/mL. The lower the IC50, the higher the capacity of extract to inhibit the production of NO. Quercetin (positive control), showed the highest NO inhibition activity compared to the extracts and fractions. The water extract had the highest NO inhibition activity compared with other extracts and fractions. The ethyl acetate fraction had the lowest NO inhibition activity.

Figure 1

Nitric oxide (NO) production level of extracts from S. spinosa leaves in LPS stimulated U937 macrophage cell line. Values are expressed as Mean ± S.E.M. a P < 0.001 b P < 0.01 c P < 0.05 versus control. Columns from left to right LPS/control (concentration mg/ml) other extracts at different concentrations AcetE: Acetone extract, MetE: Methanol extract, DcmMetE: Dichloromethane/methanol extract, AlkE: Alkaloids extract, Qur: Quercitin positive control.

Figure 2

Nitric oxide (NO) production level of extracts from S. spinosa leaves in LPS stimulated U937 macrophage cell line. Values are expressed as mean ± S.E.M. a P < 0.001 b, P < 0.01 c, P < 0.05 versus control. Columns from left to right LPS/Control, nBuF: n-Butanol fraction, WatF: Water fraction, HexF: n-hexane fraction, ChlF: Chloroform fraction, EtAcF: Ethyl acetate fraction, Qur: Quercitin positive control.

Table 1

Nitric oxide inhibitory activity and cytotoxicity of S. spinosa leaf extracts and fractions based on IC50 and LC50 values.

Samples	LC50, μg/mL)	IC50 μg/mL)
Extracts
EA	27.36±0.851	325.14±1.28a
EM	100. 91±0.03c	198.35±1.40b
ED/M	31.41±0.34h	115.62±0.98b
EAl	92. 62±0.27e	300.23±1.20a
Fractions
WF	50.00±0.08f	88.43±0.22c
nBF	98.75±0.00d	225.11±0.73a,b
EtAF	162.42±0.00a	> 750d
CF	37.38±0.08g	96.72±0.10c
HF	143.21±0.00b	300.51±2.00a
Control
Quercitin	nd	36.17 ± 1.78c
Doxorubicin	4.59±0.01j	Nd

EA: extract of acetone, EM: extract, of methanol ED/M: Extract of Dichloromethane/methanol, EAl: Extract of Alkaloids, HF: nhexane fraction, CF: Chloroform fraction, EtAF: Ethyl acetate fraction, nBF: n-Butanol fraction, WF: Water fraction. Values with different letters are significantly different at p < 0.05. nd: not determined.

Table 2

Cytotoxicity of Strychnos spinosa leaf extracts and fractions from leave on Macrophage U937 cells their selectivity index (SI).

Extracts	LC50 (μg/ml)	Selectivity Index (LC50/IC50)
AcetE	27.36±0.85	0.084
MetE	100. 91±0.03	0.508
DCM/MetE	31.41±0.34	0.271
ALKE	92. 62±0.27	0.308
Fractions
WatF	50.00±0.08	0.565
nButF	98.75±0.00	0.438
EtAcf	162.42±0.00	0.216
ChlF	37.38±0.08	0.386
HexF	143.21±0.00	0.476
Controls
Quercetin	nd	Nd
Doxorubicin	4.59±0.01	Nd

AcetE: Acetone extract, MetE: Methanol extract, DcmMetE: Dichloromethane/methanol extract, AlkE: Alkaloids extract, HexF: n- hexane fraction, ChlF: Chloroform fraction, EtAcF: Ethyl acetate fraction, nBuF: n-Butanol fraction, WatF: Water fraction, nd: not determined.

In the cytotoxicity assay, the LC50 values ranged between 27.36 and 162.42 μg/mL (Table 2). The LC50 is an indication of the toxic effect of the water, ethyl acetate and methanol fractions are 162.42, 143.21, 100.91, μg/mL respectively. Similarly, the alkaloid extract had an LC50 value of 92.62 μg/mL. The acetone and dichloromethane/methanol extracts are the most toxic among the extracts with an LC50 values of 27.36 and 31.41 μg/mL, respectively. However, the reference drug (doxorubicin) was about five to six times more toxic than the acetone and dichloromethane/methanol extracts with LC50 of 4.59 μg/mL.

It appears that more one active compound against NO production is present in these extracts because the activity did not coincide with changes in the polarity of the fractions. The chloroform fraction had a much higher activity than the ethyl acetate fraction and the ethyl acetate fraction had a higher activity than the least polar hexane fraction. The results indicated that by manipulating the extract one can change both the activity and cytotoxicity.

Discussion

The determination of anti-inflammatory activity in this work was done using LPS stimulated U937 macrophages and quantification of NO production. To ensure that the anti-inflammatory activity of the extracts and fractions were not due to cytotoxic effect of the plant; the cytotoxicity test was done on macrophages using MTT. Expression of pro-inflammatory genes (iNOS) is usually observed during inflammation caused by macrophages. The production of iNOS usually up-regulated by pro-inflammatory cytokines resulting in the production of NO from L-arginine. Agents that inhibit or stop NO production could serve as potential anti-inflammatory agents. (Oskoueian et al, 2011). The production of NO in the quercetin-treated cells used as positive control was lower than that in other samples. The water fraction had the highest anti-inflammatory activity. Phytochemical constituents such as flavonoids, phenolics, terpenoids or other constituents may be responsible for the anti-inflammatory action of the fractions and extracts of S. spinosa. Reports demonstrated that NO production and iNOS expression could be inhibited by flavonoids and other phenolic compounds in vitro. However, the degree of inhibition by flavonoids is dependent on the flavonoid sub class (Oskoueian et al, 2011).

Bacterial lipopolysaccharide or cytokines induced the production of large quantity of NO during endotoxaemia or inflammatory conditions. Inhibition of NO production that could occur through iNOS gene expression inhibition could serve as a potential therapeutic target for treating septic conditions. Curcumin, oroxylin A and wogonin are natural polyphenolic compounds that were shown to inhibit LPS-induced NO production by blocking iNOS gene expression (Oskoueian et al, 2011). Incubation of the extracts and fractions of S. spinosa with LPS prevented NO production in time and concentration dependent fashion. Plants that are used for medicinal purposes have shown activity against NO LPS-activated macrophages-induced NO production. The cytotoxicity of extracts and fractions of S. spinosa leaves was evalusted on Macrophage U937 cell lines using the MTT reduction assay. Selective toxicity against target organisms by medicinal plants is one key component in determining their clinical applications. This selective effect was usually directed at the organism or some essential metabolic pathways of the disease causing agent. Previous reports have indicated that S. spinosa had an LC50 value of 27.36 μg/mL (Isa et al 2014 a). The methanol extract of Strychnos noxvomica, a related plant had LC50 value of 18.134 mg/mL against MCF-7 cancer cell line. According to the National Cancer Institute of the United States of America, crude extracts of plants that could potentially be used as anti-cancer agent should have an LC50 less than 20μg/mL (Boik, 2001). Based on that criterion, all the extracts used in this study are considered to have relative safety which justifies their traditional use. The selectivity index (SI) of Strychnos spinosa was calculated to be 0.57. The selectivity index is a ratio that gives the relative safety and efficacy of any test substance. High value of selectivity index of a plant extract connotes that the activity is not due to a general metabolic toxin. In this study, the SI values for the extracts were less than 1 which indicates that they possess low anti-inflammatory activity in relation to their cytotoxic effect.

Conclusion

Findings of this study provide evidence for using the leaves of Strychnos spinosa in folkloric medicine for treating inflammatory-related conditions.