In vitro Screening of 5-LOX /COX-2 Dual Inhibitors from selected Medicinal plant Maytenus arbutifolia

 

Tesfay Weletnsae1, Tekleweyni Tadege1, Kidu Hintsa1, Zenebe Hagos1, Davuluri Syam Babu2,

John Dogulas Palleti3, Godavarthi Rajan Bhagyasri4, Sudhish Rai5, Krishna Chaithanya K1*,

1Department of Chemistry, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia.

2Department of Biotechnology, Vignan's Foundation for Science, Technology and Research,

Vadlamudi, 522213 Guntur, Andhra Pradesh, India.

3Research and Development, Centre for Computational and Biological Sciences,

48 -12-17, Srinagar, Near RTC Complex, Visakhapatnam - 530016, Andhra Pradesh. India.

4Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur - 515002, A. P. India.

5Department of Pharmacognosy, Tagore Institute of Pharmacy and Research, Bilaspur, Chhattisgarh, India.

*Corresponding Author E-mail: krishanchaitanyawc@gmail.com

 

ABSTRACT:

Background: The clinical management of the inflammatory diseases by using NSAIDs and selective COX-2 inhibitors, shunting arachidonic acid  pathway to the 5‐LOX pathway, which is associated with the production of high amount of Leukotrienes and cysteinyl-leukotrienes, which  are involved in pathophysiological of various type of cancers and inflammatory diseases. The substantial efforts are being made to the development of dual inhibitors against Cyclooxygenase (COX-2) and 5- Lipoxygenase (5-LOX) should be more growing demand for efficient as anti-inflammatory medicine with lower side effects than the currently available drugs. Maytenus arbutifolia (M. arbutifolia) is medicinal plant belongs to the family Celastraceae, and the leaves of M.arbutifolia have been used for the treatment of skin infections and inflammatory related disease. Objective: The current study aimed to examine the in vitro screening of M. arbutifolia leaf organic extracts for the development of anti-inflammatory agents through evaluate the inhibitory effect on Cyclooxygenase- 1and2 and 5- Lipoxygenases enzymes. Methods: The powdered dried leaves of the M. arbutifolia (100 g) was extracted with successive Soxhlet extraction by using solvents of low polarity (Petroleum ether) to a high polar solvent (Ethanol). These leaf extracts were evaluated for the in vitro anti-inflammatory activity by 5- Lipooxygenase, Cyclooxygenase-1 and Cyclooxygenase-2 inhibitory activities by ELISA method. Results: Among the tested extracts, the ethanolic leaf extracts of M.arbutifolia showed (MALET) potential 5-LOX inhibition i.e.77.58 % at 100µg/ml with IC50 of 23.76µg/ml, and also exhibited two fold COX-2 versus COX-1 inhibition, with potent COX-2 inhibitory effect with IC50 of 30.35μg/ml. The promising anti-inflammatory activity exhibited by MALET was due to the presence of secondary metabolites such as flavonoids, tannins and polyphenolic compounds. Conclusion: The current study concluded that ethanolic leaf extracts of M.arbutifolia is a promising source for isolation of 5-LOX and COX-2 dual inhibitory compound and highlights the potential traditional remedies for managing of inflammatory related diseases and cancers.

 

KEYWORDS: Maytenus arbutifolia, 5-LOX and COX-2 dual inhibitors, Anti-inflammatory agents, COX-2 Selective index.

 

 


 

INTRODUCTION: 

Inflammation is an innate immune protective response of the host is characterized by redness, swelling, heat, pain and protects against both pathogenic non-pathogenic agents and promotes healing of damaged cells and tissues1. However long-time persistence of acute inflammation can lead to chronic inflammation, results in tissue damage and failure of vital organs in the host2. During acute inflammation due to oxidative stress, genetic predispositions can be damaging the cells and tissues of the body and induce releasing of various inflammatory membrane components, which are responsible for progressing chronic inflammatory diseases3.

 

During an inflammatory response, the inflammatory stimuli such as inflammatory mediators such as Interleukin (IL) IL-1-β, IL-6, IL-8, Tumor necrosis factor-α(TNF-α), Nitric Oxide (NO), growth factors and endotoxins induce over expression of both Cyclooxygenases -2 (COX-2) and 5-Lipooxygenase (5-LOX) responsible for the synthesis of prostaglandins and leukotrienes causes progression of inflammatory diseases and different neoplasmic tumours4.

 

Several clinical studies reported that both inflammatory enzymes COX-2 and 5-LOX induce uncontrolled cell cycle progression, inhibits the apoptotic pathways, increase chemo résistance of cancer cells, stimulates angiogenesis and expression of vascular endothelial growth factor5. Both steroidal and non-steroidal anti-inflammatory drugs are used for the treatment of inflammatory-related diseases. Glucocorticoids are the steroidal hormones induce expression of nearly 130 genes responsible for providing anti-inflammation by activating phagocytosis, suppress the expression of pro-inflammatory genes Nuclear Factor kappa- β (NFk-β)6 and decreasing the consumption of ATP by cancer cells.

 

Corticosteroids are the alternative steroid hormones, inhibit the production of PGs and LTs by inactivating the phospholipase A27, and decreased the production of pro-inflammatory molecules IL-1-β, IL-6, IL-8, TNF-α and Nitric Oxide (NO)8. However, several researchers reported that a long term use of steroidal hormones responsible for side effects on metabolic pathways, glucocorticoids enhance glucose levels by degrading proteins and modulating fatty acid metabolism partly and the catabolic interference of corticosteroids leads to tissue remodelling, osteoporosis, insulin resistance and diabetes9.

 

The second categories of anti-inflammatory drug are Non Steroidal Anti-inflammatory Drugs (NSAIDs). Approximately, 60 million Americans use the non-steroidal anti-inflammatory drugs annually to treat inflammation related diseases and especially rheumatologically disorders and arthritis. NSAIDs block the production of PGs by inhibiting both COX-1 and COX-2. It is known that about 1% of chronic users of NSAIDs, such as patients with chronic inflammatory diseases develop gastrointestinal (GI) complications such as mucosal damage and bleeding10. Selective COX-2 inhibitors (COXibs) have same anti-inflammatory benefits as traditional NSAIDs with moderate effect on COX-1, but as an inhibitor of the enzyme responsible for the production of most inflammatory PGs, their drug efficacy is upheld. Furthermore, recently several reported suggested that the randomized clinical trials have shown that COXibs significantly reduce the incidence of colorectal adenomas in humans, later other clinical trials also identified an increased the risk of cardiovascular diseases. The above reports suggested that the COXibs may not be sufficiently safe for general use as cancer chemo preventive agents.

 

Recent studies suggested that both NSAIDs and selective COX‐2 inhibitors prevent the formation of PGs without modulating 5-LOX enzyme activity leads to “shunt” AA metabolism down the 5-LOX pathway, associated with the production of Leukotrienes and cysteinyl-leukotrienes. Moreover, Leukotrienes and cysteinyl-leukotrienes are pro-inflammatory and increase micro-vascular permeability and induce cell proliferation by inhibiting apoptosis11. Several COX and 5-LOX inhibitory natural products have been isolated from several plants and some of them are under various stages of preclinical and clinical evaluation for their developments as therapeutic drugs12.

 

There is clear evidence, addressing the importance of plant derived COX inhibitors in the management of the inflammatory disorders. The current strategy includes the development of dual LOX/COX inhibitors with a higher safety profile, particularly medicinal plants of folkloric use as pain relievers and anti-inflammatory agents13.

 

Maytenus arbutifolia (M. arbutifolia) is shrub medicinal plant belongs to the family Celastraceae. It is widely distributed in Africa continent such as Ethiopia, Eritrea and South Africa. In Ethiopia, it is commonly known as Atat in both Amaric and Tigrigna14. The leaves of M.arbutifolia have been used for the treatment of both bacterial and fungal pathogens responsible for causing skin infections and leaves of aqueous extracts are also used for the management of inflammatory disease.

 

Hence the present study would contribute to fill the existing gap in determining the anti- inflammatory activity of selected traditional medicinal plant M.arbutifolia in Axum city, Tigray region. Ethiopia. Therefore, the present study was to evaluate the inhibitory effect of M.arbutifolia Leaves extract on COX-I and COX-2, and 5-LOX enzymes and its phytochemical analysis.

 

MATERIALS AND METHODS:

Cyclooxygenase (COX) Inhibitor Screening Assay Kits, Lipoxygenase (5-LOX) Inhibitor Screening Assay Kits, Nordihydroguaiaretic acid (NGDA) and Indomethacin were purchased   from, Cayman Chemicals Co, USA.

 

Plant material collection and authentication:

The Leaves of M.arbutifolia were collected in 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 003 was deposited in the National Herbarium for future reference.

 

Preparation of extract and Percentage of yield:

The leaves were washed thoroughly with running tap water and one time with distilled water then the plant material was shade dried at room temperature for two weeks. The dried leaves were coarsely powdered with the electrical grinder and powdered leaf sample (100g) was subjected to successive extraction by using various polarities solvents such as petroleum ether, chloroform, acetone, and ethanol by using Soxhlet apparatus15.

 

The organic extracts were subsequently concentrated to dryness and stored desiccated at 4°C until further use. For qualitative phytochemical analysis, in vitro screening of dual COX / 5-LOX inhibitor, the extracts were placed in pre-weighed flasks before drying. The remaining plant parts residues were extracted with other solvents sequentially. Then the yield value was calculated as:

                                               Extract obtained

Percentage of Yield = ---------------------------------- ×100

                                    Total amount of plant material

 

In vitro 5-Lipoxygenase, Cyclooxygenase-1 and 2 activity:

5-Lipoxygenase inhibitoryaactivity:

According to16,17 the 5-Lipoxygenase inhibitory activity was used as an indicator of the anti-inflammatory activity. The assay was done using linoleic acid as substrate and 5-lipoxygenase as an enzyme. Inhibition studies in the presence of various concentrations of organic leaf extract of M.arbutifolia (20-100μg/ml) were recorded at 234nm using UV-Vis spectrophotometer. In the assay protocol, 160µl of 100mM sodium phosphate buffer (pH 8.0), 10μl of test samples and 20μl of soybean Lipoxygenases solution (167U/ml) were mixed and incubated at 25°C for 10 min. The reaction was initiated by the addition of 10μl of the substrate in the form of sodium linoleic acid solution. The enzymatic conversion of sodium linoleic acid to form (9Z, 11E)-(13S)-13-hydroperoxyoctadeca-9, 11-dienoate was measured by monitoring the change of absorbance at 234nm over a period of six min using UV-vis spectrophotometer. Another reaction mixture (a negative control) was prepared by replacing 10μl samples with 2.47ml mixture of sodium phosphate buffer (5ml) and DMSO (25μl) into the quartz. The percentage of inhibition was calculated by the following formula:

 

Inhibition (%) =[Ab control-Ab test / Ab control ] ×100

 

A graph was drawn with the percent inhibition as a function of the inhibitor concentration to determine the IC50 value (concentration at which there was 50% inhibition).

 

Cyclooxygenase inhibitoryactivity:

According to16,17 the in vitro COX-1 andCOX-2 inhibitory activities of organic leaf extracts of M.arbutifolia  have been evaluated using ‘COX (Ovine/Human) inhibitor screening kit’ (Cayman Chemical Company, Ann Arbor, MI) with 96-well plates. All the reagents were prepared just before use. This screening assay directly measures PGF2α produced by SnCl2reduction of COX-derived PGH2. COX-2 initial activity tubes were prepared to take 950μL of reaction buffer, 10μL of heme and 10μL of COX-2 enzymes in tubes. Similarly, COX-2 inhibitor tubes were prepared by adding 20μL of the extract at various concentrations (20-100μg/mL) in each tube in addition to the above ingredients. The background tubes corresponding to inactivated COX-2 enzymes obtained after keeping the tubes containing enzymes in boiling water for 3 min along with vehicle control. Reactions were initiated by adding 10μL of arachidonic acid in each tube and quenched with 50μL of 1 M HCl. PGH2 thus formed was reduced to PGF2α by adding 100μL of SnCl2and reading the plate at 405 nm. A graph was drawn with the percent inhibition as a function of the inhibitor concentration to determine the IC50 value (concentration at which there was 50% inhibition. The percentage inhibition was calculated by the following formula:

 

Inhibition (%) =[Ab control-Ab test / Ab control] ×100

 

Phytochemical screening:

The qualitative phytochemical analysis of Petroleum ether, chloroform, acetone, and ethanol leaf extracts of M.arbutifolia was carried out by the following methods18,19.

 

Statistical analysis:

The results were expressed as the Standard error of the Mean ± (SEM). The statistical difference between the test extracted was evaluated by One-way 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 significant difference between the extracted test group.

 

RESULTS:

The percentage yield of M.arbutifolia leaf extract:

One hundred grams of the dried leaves of M.arbutifolia were sequential extracted by using non-polar solvents from petroleum ether to polar solvent ethanol by Soxhlet’s extraction. The colour, consistency and percentage yields of these crude extracts were shown in table 1.

 

Table 1: Colour, consistency and percentage yield (w/w) of different solvent leaf extracts of M.arbutifolia

Solvent

Colour

Consistency

Percentage yield (g/100g)

Petroleum Ether

Green

Sticky

2.6

Chloroform

Dark green

Non – Sticky

3.6

Acetone

Dark green

Non – Sticky

7.8

Ethanol

Dark green

Sticky

5.6

 

As shown in the table-1 the leaves of M.arbutifolia showed the highest percentage of yield in acetone extract (7.8g/100g) and ethanol extract (5.6g/100g) followed by least percentage of yield were in petroleum ether (2.6g/100g) and chloroform (3.6g/100g) respectively. The leaves of M.arbutifolia showed the highest percentage of yield in non-polar solvent acetone, which indicated that the leaves of M.arbutifolia phytoconstituents were non-polar in nature.

 

Effect of M.arbutifolia leaf petroleum ether (MALPE), M.arbutifolia leaf chloroform (MALCH), M.arbutifolia leaf acetone (MALAC) and M.arbutifolia leaf ethanol (CFRET) on 5-Lipoxygenase (5-LOX) activity.

The percentage of 5-LOX inhibitory activity of M.arbutifolia leaf extract at the concentrations of 20-100µg/ml was shown in table-2 and figure-1. The results obtained from 5-LOX inhibitory activities of M.arbutifolia leaf extracts exhibited significant dose-dependent inhibitory activities on 5-LOX enzyme with less IC50value.

 

Table -2: Inhibitory effect of M. arbutifolia leaf extracts on 5-LOX activity

Plant extracts/Standard

Concentration

(µg/ml)

% Inhibition

IC50 (µg/ml)

MALPE

20

21.00±0.60

 

124.36

 

 

40

25.62±0.24

60

33.63±0.09

80

38.43±0.10

100

42.35±0.02

MALCH

20

36.12±0.06

94.49

40

40.21±0.04

60

43.95±0.80

80

47.51±0.90

100

51.07±0.01

MALAC

20

41.81±0.01

48.71

40

46.98±0.01

60

53.20±0.03

80

60.32±0.02

100

64.06±0.02

 

 

MALET

20

49.64±0.08

23.76

40

54.63±0.16

60

62.81±0.16

80

70.11±0.15

100

77.58±0.17

NDGA

2.5

27.19±0.45

5.54

5

44.8± 0.35

10

66.81±0.86

 

Figure 1: 5-LOX inhibitory effects of the M.arbutifolia leaf crude organic extracts (20-100 µg/ml), values are shown in mean of three replicates ± SEM.

 

The 5-LOX inhibitory activities of MALPE, MALCH, MALAC and MALET at 100µg/ml were found to be 42.35%, 51.07%, 64.06% and 77.58%, with IC50 values of 124.36µg/ml, 94.49µg/ml, 48.71µg/ml, 23.76µg/ml and 5.54µg/ml, respectively and the Nordihydroguaiaretic acid (NDGA) was taken as a positive control for comparing 5-LOX inhibitory activity of M. arbutifolia leaf extracts and its percentage of inhibition at 10µg/ml was found to be 66.81% with IC50 values of 5.54µg/ml, with significant difference i.e. (***p≤0.001 and (**p≤0.01) among all extracts, except between MALCH vs. MALAC i.e. (*p≤0.05).

 

Based on the results, it was reported that MALET showed significant 5-LOX inhibitory activity than other tested extracts.  The 5-LOX inhibitory activity of M. arbutifolia leaf extracts exhibiting the following order

MALET > MALAC> MALCH> MALPE

 

Effect of Maytenus arbutifolia leaf extracts (MALPE, MALCH, MALAC and MALET) on Cyclooxygenase (COX-1 and COX-2) activity.

The percentage of COX-1 and COX 2 enzyme inhibitory activity of M. arbutifolia leaf extracts at concentrations of 20-100µg/ml were shown in table -2. The results obtained from COX 1 and COX 2 inhibitory activities of M. arbutifolia leaf extracts revealed that MALPE, MALCH, MALAC and MALET inhibited COX-2 mediated prostaglandin synthesis with a significant IC50values compared with COX- 1 derived prostaglandin synthesis.

As shown figure-2 the COX-1 inhibitory activities of MALPE, MALCH, MALAC and MALET at 100µg/ml were found to be 23.17%, 31.81%, 44.16%, and 51.02%, with significant difference i.e. (***p≤0.001 and (**p≤0.01) among all tested extracts, with IC50 values of be 308.57µg/ml, 203.67µg//ml,123.35/ml, 92.94µg/ml and the Indomethacin was taken as a positive control for comparing the COX-1 inhibitory activity of M.arbutifolia leaf extracts and its percentage of inhibition at 10µg/ml was found to be 60.93% with IC50 7.43.µg/ml respectively.


 

Table - 2: Inhibitory effect of Inhibitory effect of M.arbutifolia leaf extracts onCOX-1 and COX-2 activities.

Plant extracts

/Standard

Concentration

(µg/ml)

% Inhibition

IC50 (µg/ml)

COX-2 Selectivity

(COX1/COX-2)

COX-1

COX -2

COX-1

COX -2

MALPE

20

13.15±0.41

17.16±0.20

 

 

 

308.57

 

 

 

215.00

 

1.44

 

 

40

16.12±0.33

22.49±0.11

60

19.75±012

23.88±0.04

80

21.42±0.01

27.98±0.02

100

23.17±0.89

30.83±0.13

MALCH

20

17.23±0.12

28.05±0.33

 

 

 

 

203.67

 

 

 

 

110.00

 

 

1.83

 

40

21.57±0.02

28.98±0.14

60

24.48±0.14

33.00±0.55

80

27.83±0.15

41.19±0.18

100

31.81±0.05

49.23±0.13

MALAC

20

27.33±0.16

39.10±0.54

 

 

 

123.35

 

 

 

80.00

1.54

 

40

31.41±0.12

42.66±0.19

60

36.73±0.14

45.05±0.10

80

41.37±0.17

48.61±0.47

100

44.16±0.13

55.49±0.82

 

 

MALET

 

20

31.45±0.12

47.91±0.04

 

 

 

 

92.94

 

 

 

 

30.35

3.06

40

36.32±0.17

52.01±0.03

60

41.08±0.38

56.57±0.06

80

48.28±047

60.82±0.95

100

51.02±0.16

65.84±0.57

Indomethacin

 

2.5

18.57±0.55

27.68±0.52

 

 

7.43

 

 

4.89

1.51

5

45.12±0.57

63.28±0.43

10

60.93±0.45

75.12±0.58

 


Figure-2: COX-1 inhibitory effects of the M.arbutifolia leaf crude organic extracts (20-100 µg/ml), values are shown in mean of three replicates ± SEM. 

The significant variable (*p≤0.05, **p≤0.01, ***p≤0.001) between M.arbutifolia leaf extracts were analysed by One way ANOVA followed by Tukey's multiple comparison test.

 

As shown figure-3, the COX-2 inhibitory activities of MFLPE, MFLCH, MFLAC and MALET at 100µg/ml were found to be 30.83%, 49.23%, 55.49%, and 65.84% respectively with IC50 values 215µg/ml, 111.36µg/ml, 80µg/ml, 30.35µg/ml significant difference i.e. (***p≤0.001 and (**p≤0.01) among all extracts, except between MALPE vs. MALCH i.e. (*p≤0.05)and the Indomethacin was taken as a positive control for comparing the COX-2 inhibitory activity of M.arbutifolia leaf extracts and its percentage of inhibition at 10µg/ml was found to be 75.12% with IC50 values 4.89µg/ml respectively.

 

Figure-3: COX-2 inhibitory effects of the M.arbutifolia leaf crude organic extracts (20-100µg/ml), values are shown in mean of three replicates ± SEM. The significant variable (*p≤0.05, **p≤0.01, ***p≤0.001) between M.arbutifolia leaf extracts were analysed by One way ANOVA followed by Tukey's multiple comparison test.

As shown in the table-2 the COX-2 selectivity (SI = IC50 COX-1/IC50 COX-2) of M.arbutifolia leaf extracts MALPE, MALCH, MALAC, MALET and Indomethacin were 1.44 1.83, 1.54, 3.06 and 1.51 respectively. The COX-2 selectivity, ratio of CFRPE, CFRCH, CFRAC, CFRET and Indomethacin were greater than (>) 1

 

Selective index of COX-1 versus COX-2 inhibition:

The COX-1/COX-2 inhibitory activities of M.arbutifolia leaf crude organic extracts (MALPE, MALCH, MALAC and MALET) and standard (Indomethacin)were evaluated using the enzyme linked immunosorbentassay (ELISA) method against ovineCOX-1and human recombinant COX-2.The half maximal inhibitory concentrations (IC50) values calculated from experimental data were shown in table -2 and figure -3. The selectivity index of M.arbutifolia leaf crude organic extracts and standard were calculated as the ratio of IC50 COX-1/IC50 COX-2. The tested M.arbutifolia leaf crude organic extracts and standard showed different potential of COX-1/2 inhibition.

 

M.arbutifolia leaf ethanol extract (MALET), showed approximately twofold COX-2 versus COX-1 inhibition, with potent COX-2 inhibitory effect with IC50of 30.35μg/ml, and also showed twofold COX-2 selectivity than the standard from the table- 2, it is revealed that no tested extracts of M.arbutifolia leaf showedCOX-1 selective inhibitory activity.

 

Figure- 4: The percentage inhibition of the COX-1/COX-2 enzymes and Selective index of COX-1 versus COX-2 inhibition by M.arbutifolia leaf, organic extracts (20-100 µg/ml)

 

Based on the results, it was observed that MALET showed significant COX-2 selectivity inhibitory activity than other tested extracts. The COX-2 selectivity ratio of M.arbutifolia leaf extracts exhibit the following order.

MALET > MALAC> MALCH> MALPE

 

The qualitative phytochemical analysis of leaves extract of M.arbutifolia:

The qualitative phytochemical analysis of petroleum ether, chloroform, acetone and ethanol leaves extract of M. arbutifolia were showed in the table -3, all the tested four leaves extract of M.arbutifolia contained Alkaloids, Flavonoids, Terpenoids and Coumarins.

 

As shown in the table-3, the petroleum ether leaf extract of M.arbutifolia showed that presence of Alkaloids, Flavonoids, Tannin and Phenolic compounds, Quinones, and remaining primary and secondary metabolites were absent. The chloroform leaf extract of M.arbutifolia was shown the presence of both primary and secondary metabolites except Amino acid and Protein, Volatile oils; Terpenoids, Quinones, Steroids and Anthraqunones were showing absent. The acetone leaf extract of M. arbutifolia showed the presence all the secondary metabolites except volatile oils, steroid and Anthraquinones were absent. The ethanolic leaf extracts of M. arbutifolia showed the presence all the secondary metabolites except volatile oils, steroids and Anthraquinones were showing absent.

 

From the table-1 and3, it was concluded that the highest percentage of yield was registered in acetone extract (7.8g/100g) and but maximum number of secondary metabolites were found in ethanolic leaf extracts of M.arbutifolia.

 

Table - 3: Qualitative phytochemical analysis of different organic solvent crude leaves extracts of M.arbutifolia.

Phytoconstituents

Solvent

Petroleum ether

Chloroform

Acetone

Ethanol

Alkaloids

+

+

++

++

Flavonoids

+

+

++

++

Tannins/ Phenols

+

+

++

+++

Cardiac Glycosides

-

+++

++

+

Terpenoids

-

-

++

+

Saponins

-

-

++

+++

Steroids

-

-

+

-

Quinones

+

-

+

++

Coumarins.

-

+

+

++

Carbohydrates

-

+

++

++

Volatile  oils

+

-

-

-

Anthraquinones

-

-

-

-

Amino acid and protein

-

-

+

++

“+” indicates presence of primary / secondary metabolites.

“-” indicates presence of primary / secondary metabolites.

 

DISCUSSION:

The ethanolic leaf extracts of M.arbutifolia (MALET) showed potential 5-LOX inhibitory activity of i.e. 77.58 % at 100µg/ml with IC50 of 23.76µg/ml, which was relatively similar to standard Nordihydroguaiaretic acid (NDGA) 66.81 with IC5O of 5.54µg/ml) as shown in the table-2, the 5-LOXinhibitory activity was, attributed to the anti-inflammatory potential of the ethanolic leaf extracts of M.arbutifolia. Yarla et.al.,20 reported that methanolic seed coat extract of (pericarp) Flabellifer and its isolated dammarane triterpenoid showed excellent 5-LOX inhibitory activity with IC50 of 21.4±1.45μg/ml, 9.7±0.02μM respectively and also from the in silico studies of dammarane triterpenoid on 5-LOX enzyme, the bioactive compound dammarane triterpenoid-1 showed strong binding affinity on active site amino acids of 5-LOX , responsible for inhibition of enzyme activity.

 

The 5-LOX inhibitory activity of ethanolic leaf extracts of M. Arbutifolia (MALET) due to the presence of flavonoids, tannin and phenolic Compounds, terpenoid, Quinones and Coumarins. There was positive correlation observed between the 5-LOX inhibitory activities of leaf extracts of M.arbutifolia and the considerably high amounts of secondary metabolites are present such as flavonoids, tannin and phenolic compounds terpenoids.

 

In the present study, the various concentrations (20-100µg) of organic leaf extracts of M.arbutifolia, was screened for its inhibitory efficacy against COX-1 and COX-2. The in vitro cyclooxygenase inhibitory study demonstrated that the M.arbutifolia leaf extracts MALPE, MALCH and MALAC showed twofold selective inhibitory activities against COX-2 as compared to COX-1 and MALET showed three fold selective inhibitory activities against COX-2as compared to COX- 1.Bharat Reddy et.al.,21 reported that the ethanolic extract of fruit coat powder of Terminalia chebula showed, inhibitory activity on COX-1 and COX-2 enzymes with IC50 of 90 and 3.75μg/ml, it was clearly evident that ethanolic fraction of fruit coat powder of Terminalia chebula inhibits COX-2 and COX-1 with 25 fold preference towards COX-2.

 

The results of the present study indicated that MALET shown selective index (3.06) and inhibited COX-2 catalyzed prostaglandin synthesis with an IC50 value of 30.35µg/ml, compared with COX- 1 derived prostaglandin synthesis which showed an IC50 value of 92.94μg/ml. At the same time, CX-2 selective index of MALET was twofold more than to the standard NSAIDs such as Indomethacin (1.51). A low COX-1/COX-2 ratio indicates a preferential COX-2 inhibitor, which is pharmacologically valuable.

 

The results of the present study revealed that the dual inhibitory activity of organic leaf extracts of M.arbutifoliaon5-LOX/COX enzymes were found to be due to the presence of phytocompounds as shown in table-3, such as flavonoids, Tannins/ Phenols, terpenoids, coumarins and quinones. Among the all tested leave extracts of M. arbutifolia, the ethanolic leaf extract shown significant 5-LOX and COX dual inhibitory activities, due to the presence of maximum number of secondary metabolites, such as flavonoids, tannins/ phenols, terpenoids.

 

Shang et.al.,22 reported that Alkaloids, from A. Scholaris leaves showed anti-inflammatory and analgesic activity by inhibiting, COX-1, COX-2 and 5-LOX.  Khan et.al., 23 reported that Morinda citrifolia Linn, leaf organic extracts showed inhibitory effect on COX-2 and 5-LOX enzymes.

 

CONCLUSION:

All organic extracts of M.arbutifolia leaves (MALPE, MALAC, MALCH and MALET) showed considerable significant 5-LOX and COX-2 inhibitory activity with less IC50values among the all tested organic extracts. The ethanolic leaf extract of M.arbutifolia (MALET), showed twofold COX-2 versus COX-1 inhibition, with potent COX-2 inhibitory effect with IC50 of 30.35μg/ml, and also shown twofold COX-2 selectivity than the standard. The most promising anti-inflammatory activity exhibited by MALET among the other solvent extracts, due to the presence of secondary metabolites such as flavonoids, terpenoid, tannins and polyphenolic compounds. The result suggested that the ethanolic leaf extracts of M.arbutifolia (MALET) showed potential anti-inflammatory activity by exhibiting inhibitory activity on both 5-LOX and COX-2 enzymes and confirms traditionally reported anti-inflammatory capacity of M.arbutifolia. Hence, further investigations will be carried out on leaf extracts M.arbutifolia (MALET) to isolate, identify and characterized the anti-inflammatory compounds (s) which is responsible for 5-LOX and COX-2 inhibitory activity by activity guided fractionation.

 

ACKNOWLEDGMENTS:

The authors sincerely thanks to the Department of Chemistry, Aksum University, Axum, Ethiopia for providing laboratory facilities.

 

FINANCIAL SUPPORT:

This project work was funded by Ministry of Education, Ethiopia in the form Student Project work.

 

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

 
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Received on 02.08.2020           Modified on 24.10.2021

Accepted on 16.06.2022          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):241-248.

DOI: 10.52711/0974-360X.2024.00038