The medicinal plant Calpurnia aurea leaves act as an anti-in(cid:976)lammatory source of 5-Lipoxygenase / Cyclooxygenase-2 dual inhibitors

g/ml and the C.aurea leaves acetone extract showed the selective COX-2 index (2.35) inhibitory activity with an IC 50 of 200.75 (cid:22) g/ml. The current work revealed that the C. aurea leaf acetone extract (CALAC) is shows potential source for the isolation and development of 5-LOX and COX-2 dual inhibitory compounds for management of in(cid:976)lammatory related diseases and in(cid:976)lamma-tory cancers.

Calpurnia aurea, Cyclooxygenase, 5-Lipoxygenase, Anti-in lammatory activity A Calpurnia aurea is a medicinal plant belonging to the family of Fabaceae. Traditionally in Ethiopia the leaves have been used for the treatment of diarrhea, leishmaniasis, swelling, cough and snake bite. Based on the traditional use the leaves of C. aurea might be a promising source for development of anti-in lammatory agents. The objective of the current research was to explore the 5-Lipooxygenase (5-LOX), Cyclooxygenase-1 & Cyclooxygenase-2 (COX-1 & COX-2) inhibitory activity of in lammatory enzymes of organic leaves crude extract of C. aurea and its phytochemical analysis. The dried leaves powder of the C. aurea (100 g) was extracted with successive soxhlet extraction by solvents of low polarity (Petroleum ether) to a high polar solvent (Ethanol). These organic leaf extracts C. aurea were assessed for the in vitro anti-in lammatory activity by 5-Lipooxygenase, Cyclooxygenase-1 & Cyclooxygenase-2 inhibitory activities by ELISA method and further subjected to phytochemical screening. The ethanolic leaves extract of C.aurea showed signi icant 5-LOX inhibitory activity of 51.07% at 100 µg/ml with IC 50 of 95.30µg/ml and the C.aurea leaves acetone extract showed the selective COX-2 index (2.35) inhibitory activity with an IC 50 of 200.75µg/ml. The current work revealed that the C. aurea leaf acetone extract (CALAC) is shows potential source for the isolation and development of 5-LOX and COX-2 dual inhibitory compounds for management of in lammatory related diseases and in lammatory cancers.

INTRODUCTION
In lammation is an innate immune protective response of vascular tissues and innate immune cells against infection and to restore tissue injury. Dysregulation of acute in lammation leads to chronic in lammatory-related diseases such as diabetes, obesity, arthritis and inducing malignant celltransformation in different types of cancers (Kim et al., 2018;Clark et al., 2017). The Arachidonic acid (AA) is a 20 carbon polyunsaturated fatty acid extensively present in mammalian membrane systems involved in different in lammatory pathways (Sevanian and Kim, 1985) and A. A act as precursor for three major in lammatory pathways by both prostaglandins and leukotrienes synthesis by cytochrome P-450 monooxygenase (Montellano and Paul, 2015), 5-Lipoxygenase (Piomelli et al., 1987) and Cyclooxygenase (Santos et al., 2017) pathways.
During in lammation, the 5-Lipoxgenase (5-LOX) activation leads to the active synthesis of the Leukotrienes (LTs) such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and LTC4, LTD4, and LTE4 are known as "cysteinyl leukotrienes" implicated in the pathogenesis of various in lammatory diseases, asthma, progression of tumors (Epstein et al., 1990). Cyclooxygenases (COX) are prostaglandin-endoperoxide synthase (E.C. 1.14.99.1) act on arachidonic acid (AA) and produce different types of effective proin lammatory prostaglandins such as PGE 2 and PGF 2a (Shaikh et al., 2015). In mammalian cells, COX exists as COX-1 and COX-2 isoforms, these are close in their structure around 60-65% of the sequence are nucleotide sequences are conserved (Hwang et al., 2010). COX-1 is a house keeping enzyme that expressed all cell types of mammalian tissues such as vascular endothelium, platelets stomach, kidney, and uterine epithelium and maintained the normal physiological functions (Samuelsson et al., 2010).
Several clinical and molecular reports on chronic in lammatory diseases and cancers proven that the Arachidonic Acid metabolites are up-regulated and plays a vital role in Auto-in lammation and tumorigenesis. Pharmacological inhibition of the Archadonic Acid metabolic in lammatory pathway by natural as well as synthetic molecules have been considered as novel approaches to the advancement of dual 5-LOX and COX-2 inhibitors without any side effects for treating various in lammatory disorders and cancer (Weletnsae et al., 2019).
The NSAIDs are anti-in lammatory drugs, but nonspeci ic inhibition of COX-1 and long term administration causes severe side effects such as gastrointestinal (GI) ulceration, obstruction, and has restricted the therapeutic usage of NSAIDs (Harirforoosh et al., 2013). Conventional NSAIDs is being replaced by selective COX-2 inhibitors, such as Celecoxib, suggestive for in lammation and cancer. The slecive inhibition of COX-2 enzyme "shunt" the A.
A metabolism pathway towards the 5-LOX pathway, which can induce production excess production of Leukotrienes and cysteinyl-leukotrienes. These Leukotrienes and cysteinyl-leukotrienes are acting as pro-in lammatory increase micro-vascular permeability and induce cell proliferation by inhibiting apoptosis (Ziboh et al., 2004).
Plant derived natural products act as cancer preventive and anti-in lammatory agents by targeting COX-2 and 5-LOX, for reducing both gastric and cardiovascular side effects by balancing AA metabolism in the body (García-Lafuente et al., 2009). Hence, researchers are considering an alternative for synthetic dual 5-LOX and COX-2 inhibitor from medicinal plants.
Calpurnia aurea is belongs to the family of Fabaceae and well known medicinal plant in Axum town, Tigray region, Ethiopia. This lowering plant commonly known in Amaric Digita and Hitsawts in Tigrigna. This plant species are extensively disseminated in Africa and Southern Asia (Asres et al., 1986). Literature survey brings to light that, the leaf and stem of C.aurea have been used for treatment of human and live stock infectious and non-infectious disease (Jäger et al., 1986).
Since long time in Ethiopia the leave of C. aurea is used for the treatment of infectious protozoan, bacterial, fungal and viral diseases such as syphilis, malaria, leishmaniasis, elephantiasis, diarrhoea, trachoma, rabies and non-infectious diseases such as diabetes, hypertension, different swellings, stomach-ache, bowel, and bladder disorders (Umer et al., 2013). Therefore, current research focused on investigated inhibitory effects of the organic leaf extracts C. aurea on 5-LOX and COX-I & COX-2 in lammatory enzymes and its phytochemical analysis.

Study area
The leaves of selected Medicinal plant C. aurea were collected from Axum city, in the Central Zone of region Tigray, Ethiopia. It has an elevation of 2,131 meters (6,991 ft) above sea level latitude of 14 • 07' 15.92" N longitude 38 • 43' 24.13". The average annual temperature in Axum is 18.3 • C and the rainfall averages is 652 mm.

Plant material collection and authentication
The Leaves of C. aurea, were collected in Axum city, Central Zone of Tigray region. The plant was authenticated by the Department of Biology, National Herbarium, Addis Ababa University, and voucher specimen number of TW 002 was deposited in the National Herbarium for future reference.

Preparation of extract and Percentage of yield
The leaves were rinsed carefully with running tap water, and one time with distilled water, then the leaves were shade dried for 15 days. The dried leaves were coarsely powdered with the electrical grinder and powdered leaves of C. aurea (100 g) was taken for successive extraction by using various polarities solvents such as petroleum ether, chloroform, acetone, and ethanol by using soxhlet apparatus (Das et al., 2010).
The organic leaf extracts of C. aurea were subsequently concentrated to dryness and stored desiccated at 4 • C until further use for qualitative phytochemical analysis, In vitro screening of dual 5-LOX/ COX-2 inhibitors, the extracts were placed in preweighed lasks before drying. The remaining plant parts residues were extracted with other solvents sequentially. Then the yield value was calculated as Percentage of Yield =

Extract obtained T otal amount of plant material
× 100
According to modi ied method of (Bisht et al., 2014;Kumar et al., 2016) 160µl of 100mM sodium phosphate buffer (pH 8.0), 10µl of tested leaf extracts C. aurea and 20µl of soybean Lipoxygenases solution (167 U/ml) were mixed and allowed to incubated at 25 • C for 10 min. The reaction was then started by the addition of 10µl of the ω-6 fatty acids unsaturated fatty acid linoleic acid as a substrate in the form of sodium linoleic acid solution.
The 5-LOX enzymatic change of sodium linoleic acid to (9Z, 11E)-(13S)-13 hydroperoxyoctadeca-9, 11dienoate was measured by monitoring the change of absorbance at 234 nm over a period of six min using UV-Visible spectrophotometer. Negative control was prepared by using 2.47 ml mixture of sodium phosphate buffer (5 ml) and DMSO (25µl) into the quartz. IC 50 values of tested plant extracts were determined by using graph pad prism Graph pad prism 6.0 software.
A graph was drawn with the Percent Inhibition as a function of the inhibitor concentration to determine the IC 50 value (concentration at which there was 50% inhibition).
According to modi ied method of (Bisht et al., 2014;Kumar et al., 2016) COX-I & COX-II initial and inhibitory tubes were prepared by taking 950µL of reaction buffer, 10µL of heme and COX-I and COX-2 enzymes in the tubes. In inhibitor tubes 20µL of the tested plant extracts at various concentrations (20-100µg/mL) in each tube in addition to the above ingredients. The COX-I & COX-II enzymatic reactions were initiated by adding 10µL of arachidonic acid in each tube and quenched with 50µL of 1 M HCl. PGH 2 thus formed was reduced to PGF2α by adding 100µL of SnCl 2 , and reading the plate at 405 nm. IC 50 values of tested plant extracts were determined by using graph pad prism Graph pad prism 6.0 software.

Phytochemical analysis
The phytochemical analysis of tested extracts were carried out by using the standard methods (Audu et al., 2007).

Statistical analysis
The results were represented as the standard error of the mean ± (SEM). The statistical difference between the tested extracts were analyzed by oneway analysis of variance followed by Turkey 'multiple comparison test Graph pad prism 6.0 software and followed by Dunnett's t-test.*p≤0.05, **p≤0.01, ***p≤0.001 represents a signi icant difference between the extracted test group.

Percentage yield
One hundred grams of the dried leaves of C. aurea were extracted with petroleum ether, chloroform, acetone and ethanol solvent by Soxhlet's extraction. The colour, consistency and obtained percentage yields of these crude extracts were shown in Table 1.
As presented in Table 1 the leaves of C. aurea reported the highest percentage of yield in acetone extract (6.1g/100g) and ethanol extract (5.7g/100g) followed by least percentage of yield  The results obtained from 5-LOX inhibitory activities of C.aurea leaf extracts exhibiting dose-dependent inhibitory activities on 5-LOX enzyme, with lowest IC 50 value were in petroleum ether (1.4g/100g) and chloroform(1.7g/100g) respectively. The leaves of C.aurea showed the highest percentage of yield in non-polar solvent acetone, which indicated that the leaves of C.aurea phytoconstituents were non-polar in nature.
As shown in Figure 1, the 5-LOX inhibitory activities of CALPE, CALCH, CALAC and CALET at 100µg/ml were found to be 20.46%, 38.97%, 47.69% and 51.07% respectively, with signi icant difference, i.e. (***p≤0.001 and (**p≤0.01) among all extracts, except between CALAC, CALET i.e. (*p≤0.05) and the Nordihydroguaiareticacid (NDGA) was taken as a positive control and % of inhibition at 10µg/ml was found to be 66.81%. The IC 50 values of CALPE, CALCH, CALAC, CALET and NDGA were shown in    Based on the results, it was observed that CALET showed signi icant 5-LOX inhibitory activity than other extracts. The 5-LOX inhibitory activity of C.aurea leaf extracts exhibiting the following order,

Effect of C. aurea leaf extracts (CALPE, CALCH, CALAC and CALET) o Cyclooxygenase (COX-1 and COX-2) activity
The percentage of COX 1 and COX 2 enzyme inhibitory activity of C. aurea leaf extracts at concentrations of 20, 40, 60, 80and 100µg/ml were shown in Table 3. The results obtained from the COX 1 and COX 2 inhibitory activities of C. aurea leaf extracts revealed that CALPE, CALCH, CALAC and CALET inhibited COX-2 mediated prostaglandin biosynthesis with a signi icant IC 50 values, compared with COX-1 derived prostaglandin synthesis.

Selective index of COX-1 versus COX-2 inhibition
The COX-1/2 inhibitory activities of C. aurea leaf crude organic extracts (CALPE, CALCH, CALAC and CALET) and standard (Indomethacin) were evaluated using the enzyme linked immunosorbent assay (ELISA) method against ovine COX-1 and human recombinant COX-2. As showed in the Table 3 and Figure 4 the selectivity index of C. aurea leaf crude organic extracts and standard were calculated as the ratio of IC 50 COX-1/IC 50 COX-2. The tested C. aurea leaf crude organic extracts and standard showed the different potential of COX-1/2 inhibition. C.aurea leaf petroleum ether extract, chloroform extract, ethanolic extract, shown COX-1inhibition and C.aurea leaf acetone extract (CALAC) shown twofold COX-2 inhibition, with potent COX-2 inhibitory effect with IC 50 of 200.75µg/ml. The chief capable 5-LOX and COX-2 dual inhibitors found to be in CALAC, which has a signi icant point of selectivity towards COX-2, and shown twofold COX-2 selectivity than the standard. FromTable 3, it was revealed that (CALPE, CALCH, and CALET) shown COX-1 selective inhibitory activity.
Based on the results, it was revealed that CALAC exhibited signi icant COX-2 selectivity inhibitory activity than other tested extracts and Indomethacin. The COX-2 selectivity ratio of C aurea leaf extracts exhibiting the following order,

CALPE <CALCH<CALET<CALAC
The qualitative phytochemical analysis of petroleum ether, chloroform, acetone and ethanol leaves extract of C. aurea The qualitative analysis of phytochemicals present petroleum ether, chloroform, acetone, and ethanol leaves extract of C.aurea was presented in Table 4, and the result showed that all the four leaves extract of C. aurea contained alkaloids, tannin and phenolic compounds. The petroleum ether leaves extract of C.aurea was screening its phytochemical constituents as shown in Table 4, the result indicated that petroleum ether extracts shown the presence of alkaloids, terpenoid, quinones and remaining both primary and secondary metabolites were showing absent. The chloroform leaves extract of C.aurea was screening its phytochemical constituents as shown in Table 4, the result indicated that the presence of primary metabolites amino acids, proteins and secondary metabolites such as alkaloids, lavonoids, terpenoid, tannin and phenolic compounds, and coumarins, remaining phytoconstituents were showing absent.
The acetone leaves extract of C. aurea was screening its phytochemical constituents as shown in Table 4, the result indicated that the presence of alkaloids, lavonoids, saponins, tannin and phenolic compounds, terpenoids, coumarins, amino acids and proteins, remaining phytoconstituents were showing absent. The ethanolic leaves extract of C.aurea was screening its Phytochemical constituents as shown in Table 4. The result indicated, all primary and secondary metabolites such as carbohydrates, amino acids and proteins and alkaloids, lavonoids, saponins, tannin and phenolic compounds, glycosides, terpenoids, quinones, coumarins, the remaining anthraqunones and volatile oils were showing absent.
From Tables 1 and 4 it was concluded that the highest percentage of yield was registered in ethanol extract (4.4g /100g) and maximum number of secondary metabolites were found in ethanolic leaf extracts of C.aurea.
Among all tested organic extracts, ethanolic leaf extract of C.aurea displayed, potent 5-LOX inhibition i.e. 51.07 % at 100 µg/ml with (IC 50 = 95.30µg/ml), which was relatively similar to standard NDGA 66.81 with (IC 50 = 5.54 µg/ml) as shown in the Table 2, the 5-LOX inhibitory activity was credited to the antiin lammatory potential of the ethanolic leaf extract of C.aurea. (Akula and Odhav, 2013) reported that among the eighteen selected plants studied against 5-LOX inhibitory activity, Bidenspilosa is a lowering plant belongs to family aster and native to America (IC 50 = 21.8 µg/ml) showed highest anti-in lammatory activity while Emex australis indigenous medicinal plant of South Africa belongs to the family Polygonaceae (IC 50 81.4 µg/ml) showed least inhibition of 5-LOX activity and they concluded that the anti-in lammatory activities of Biden spilosa could be explained by the potent inhibitory effects of their phenolic compounds on arachidonic acid metabolism through the lipoxygenase pathway.
The 5-LOX inhibitory activity of ethanolic leaf extracts of C.aurea (CALET) owing of lavonoids, tannin and phenolic compounds, terpenoid, quinones and coumarins. It is possible correlation observed between the anti-in lammatory activities of the studied plant was extracts is due to the considerably high amounts of phenolic compounds. From in vitro cyclooxygenase inhibitory study con irmed that the C.aurea leaf acetone extracts (CALAC) showed twofold selective inhibitory activities against COX-2 as compared to COX-1, than other organic leaf extract of C.aurea (CALPE, CALCH, and CALET) and (Burnett et al., 2007) reported that Baicalensis extract and isolated baicalin showed about twofold more COX-2 versus COX-1 inhibition when IC 50 values were compared.
The current work indicated that CALAC shown selective index (2.35) and inhibited COX-2 mediated synthesis of PGE 2 derivatives with an IC 50 value of 200.75µg/ml, compared with COX-1 with an IC 50 value of 472.88µg/ml.
At the same time, the COX-2 selective index of CALAC was twofold to the standard Indomethacin (1.51). A low COX-1/COX-2 ratio speci ied a preferential COX-2 inhibitor, which is pharmacologically valuable. The COX-2 selectivity, ratio of CALAC and Indomethacin were greater than (>) 1 but CALPE, CALCH CALET shown less than (< 1).
Present results revealed that the dual inhibitory activity of organic leaf extracts of C.aurea on 5-LOX/COX enzymes was found to be the presence of phytocompounds such as lavonoids, tannins/ phenols, terpenoids, coumarins, and quinones. Among the all tested plant extracts of C.aurea, leaves acetone extract shown signi icant 5-LOX and COX dual inhibitory activities, due to the presence of a highest number of secondary metabolites, such as lavonoids, tannins/ phenols, terpenoids.
Our results also correlated with other antiin lammatory work of medicinal plants Catechin, a natural lavonoid isolated from Acacia catechu L, shown in vitro COX-2 and 5-LOX inhibitory activity (Burnett et al., 2007) and also inhibited both ovine COX-1 and COX-2 at IC 50 of 15mg/ml, by decreased production of PGE 2 .

CONCLUSIONS
Based on the results it was concluded that the C.aurea leaf ethanolic extract of (CALET) con irmed the promising 5-LOX inhibitory activity which is comparable to standard NDGA. The C.aurea leaf acetone extract (CALAC) showed promising 5-LOX and COX-2 selectivity inhibitory activity than other tested extracts and standard NDGA & indomethacin. From the current study the C. aurea leaf acetone extract is a potential source for the isolation and development of 5-LOX and COX-2 dual inhibitory compounds for the management of in lammatoryassociated diseases.