J Med Allied Sci 2019; 9(1):19-25 DOI: 10.5455/jmas.23465

Original article

Phytochemical evaluation, antioxidant and COX-1/COX-2 inhibitory activities of the methanol extract of the leaf of Cochlospermum planchonii Hook. F. (Cochlospermaceae)

Paul Ngbede Olotu1, Abubakar Ahmed2, Oluyemisi Folashade Kunle3, Ijeoma Adanma Olotu4, Francis Maji Agwom5, Ukpe Ajima5

Affiliation(s):

1Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, University of Jos, Jos-930001, Plateau State, Nigeria.

2Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Kaduna State, Nigeria.

3Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD), Garki, Abuja, Nigeria.

4Department of Biochemistry, Faculty of Medical Sciences, University of Jos, Jos-930001, Plateau State, Nigeria.

5Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, Jos-930001, Plateau State, Nigeria.

Corresponding author: Dr. Paul Ngbede Olotu, Lecturer, Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, University of Jos, PMB 2084, Jos-930001, Plateau State, Nigeria.

Phone: +234-8035939772 Email: olotupauln@yahoo.com

Abstract

Cochlospermum planchonii is widely used in folk medicine in many African countries and has been shown to have beneficial activity in a number of disease conditions. The plant is proposed to contain anti-inflammatory compounds that can relieve pain and inflammation associated with elevated levels of prostaglandins in the body. The present study was therefore aimed at investigating the phytochemicals present in the methanolic extract of the leaves of Cochlospermum planchonii L. (Cochlospermaceae) and also evaluating the antioxidant and COX-1/COX-2 inhibitory activities of the extract. Antioxidant activity was determined using a 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. Phytochemical evaluation performed using standard known methods revealed the presence of steroids/terpenes, cardiac glycosides, saponins, tannins, flavonoids and alkaloids. The extract had significant antioxidant activity of 93±0.11% in the DPPH assay. The extract was found to inhibit COX-2 selectively. The preliminary antioxidant screening indicated that the extract possesses the potential to scavenge free radicals and the capacity to bleach DPPH. The COX assay was carried out with a view to providing a simple mechanism by which the extract exhibits its anti-inflammatory action. The present results are consistent with the traditional use of this plant in phytotherapeutic preparations for managing pain and inflammation related ailments.                   

Key words: Cyclo-oxygenase inhibition, DPPH, Ethnopharmacology, Free radicals, Secondary metabolites

Running title: Studies on the leaf extract of Cochlospermum planchonii Hook. F

Introduction

Cochlospermum planchonii is a low shrubby savanna plant that can grow as high as 2.5 meters and reproduces from the seeds and rhizomes. Among the tribes of Nigeria, it is called “N′ Dribala” (Fulani), “Rawaya” or “Kyamba” (Hausa), “Abanzi” (Igbo) and “Gbehutu or Feru” (Yoruba). The plant is commonly used as a traditional medicine in several sub-Saharan African countries such as Cote d’Ivoire, Nigeria, Burkina Faso, Ghana, Cameroon, Guinea, Gambia and Senegal1. It is also a common weed of cultivated fields in the Sudan and Guinea savannah zones2. The leaf, stem and root bark of C. planchonii have all demonstrated strong antifungal activity against Colletotrichum capsici3. The aqueous leaf extract of the plant has been shown to have blood glucose lowering and antidyslipidemic potential4 while the essential oil of the plant has been shown to possess antibacterial activity5. The decoction of the plant leaves has also been used as an alternative therapy for diarrhoea6, gonorrhoea, jaundice and gastrointestinal disease7. Analysis of the root extract showed the presence of egallic acid, zinc salt and manganese8.

Reactive oxygen species (ROS) or sometimes reactive nitrogen species (RNS) are highly reactive entities containing an unpaired electron. Some prominent examples of these species include superoxide anion (O2.-), hydrogen peroxide (H2O2), peroxyl radical (HO), singlet oxygen (1O2) and peroxynitrite (ONOO-). They are known to cause damage to human cells9. To neutralize the assault of these ROS, living cells have a biological defense mechanism made up of enzymatic antioxidants that convert reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as O2•‾ and NO• to harmless species. However, no enzymatic action is known to scavenge radicals such as ROO, OH, 1O2, and ONOO‾. Therefore, the burden of defense relies on a variety of non-enzymatic antioxidants such as vitamin C and E and many phytochemicals that have the property of scavenging oxidants and free radicals10. An antioxidant is any substance that, when present at low concentrations, compared with those of an oxidizable substrate (lipids or proteins, etc.) significantly delays or prevents oxidation of that substrate. It can be broadly classified as enzymatic such as superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) and their precursors or non-enzymatic such as vitamins from fruits, polyphenols from vegetables and medicinal plants. Antioxidant protection may be based on several mechanisms of action, which include:  scavenging capacity against ROS/RNS, reducing capacity, metal chelating capacity, anti-oxidative enzymes e.g. nitric oxide synthase, xanthine oxidase and cyclooxygenase11. A number of phytochemicals obtained from plants have been shown to possess antioxidant activity and can serve as lead compounds for the development of new drugs12.

Inflammation is a response that occurs in living tissues to injury, infection or irritation and is characterized by pain, swelling, redness and heat. Inflammatory abnormalities are a large group of disorders that underlie a wide variety of human diseases such as hay fever, cancer, ischemic heart disease, rheumatoid arthritis, etc13. The term eicosanoid covers biologically active lipid mediators (C20 fatty acids and their metabolites) including prostaglandins, thromboxanes, leukotrienes and other oxygenated derivatives, which are primarily produced by three classes of enzymes; cyclooxygenases (COX), lipoxygenases (LOX) and cytochrome P450 mono-oxygenases. The immediate precursor of the eicosanoids is arachidonic acid which is hydrolyzed from phospholipids by Phospholipids A2, also a membrane component14. Eicosanoids are implicated in pains, inflammatory conditions15. Cyclooxygenases (COX) is an enzyme that catalyzes the conversion of arachidonic acid (AA) into a large number of eicosanoids with potent biological effects14.

Cochlospermum planchonii L. (Cochlospermaceae) leaf is locally used as medicine without any scientific validation. The chemistry of Cochlospermaceae is poorly known16.  In the present study, the methanolic extracts of the leaves of Cochlospermum planchonii was therefore chosen to validate its traditional use in folk medicine.  

Materials and methods

Plant collection, identification and preparation

Cochlospermum planchonii leaf was collected on the 9th June 2012 from ‘Babare’ locality, in Jos North Local Government Area of Plateau state, Nigeria. The plant was identified in the field using the pharmacognostic descriptions and keys. The identity of the plant was authenticated at the Department of Horticulture and Landscape Technology, Federal College of Forestry, Jos, Nigeria, and assigned Voucher specimen Number (FHJ 1011). The plant was collected and air dried at room temperature under shade until a constant weight was obtained for a period of three weeks. The plant was then pounded to powder using a pestle and mortar, sieved with a mesh of size-20 and stored in an air-tight container until when required for use.

Chemicals and reagents

All the solvents used in the study were of analytical grade.

Equipment

UV-Visible double beam spectrophotometer Model UV-1620PC (Shimadzu, Japan), Beckman Model LS 6000LL Liquid Scintillation Counter (Beckman Instruments, Palo Alto, United States of America), Rotary Evaporator Rotavapor R-300 (BUCHI, Switzerland).

Plant extraction

The powdered drug (1 kg) was extracted with methanol (3 Liters) by maceration/electric shaking for 72 hours. The extract was concentrated under reduced pressure using a Rotary evaporator to obtain the methanol extract (18.40% yield).

Phytochemical screening

The presences of phytochemical constituents of the different extracts were investigated using standard methods17.

Thin Layer Chromatographic (TLC) analysis

The TLC profile of the methanol extract was obtained by spotting the extract on the TLC plate and developing it in Ethyl acetate: Methanol: Hexane (7:7:3). The plates were sprayed with anisaldehyde/ sulphuric acid as detecting reagent and heated at 110°C. The chromatogram was scanned using a previously described method12.

Antioxidant assay                                                                          

It was carried out on pre-coated silica thin layer chromatography (TLC) plate and appropriate solvent systems. The 2, 2-Diphenyl-l-Picrylhydraxyl (DPPH) reagent was used as detecting reagent. A yellow spot against purple background would suggest presence of free radical scavenging compounds. The antioxidant activity (free radical scavenging activity) of aqueous methanolic extract on the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was determined according to the method described by (Brand-Williams et al, 1995). 12.5 mg of the extract was dissolved in methanol using 25 mL volumetric flask. The following concentrations of aqueous methanolic extract were prepared 500, 250, 125, 62.50, 31.25, 15.62, 7.8125, 3.91, 1.95, 0.98, 0.49, 0.245, 0.1225; 0.06125 μg/mL.  All the solutions were prepared with methanol as solvent. About 2 mL of each prepared concentration was mixed with 4 mL of 50 μM DPPH solution in methanol. The experiment was done in triplicate. The mixture was vortexed for 10 seconds to homogenize the mixture and test tubes were incubated for 30 mins at room temperature in the dark. After 30 minutes of incubation, the absorbance was measured at 515 nm on a UV-Vis spectrophotometer.

Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Gallic acid, vitamin C and rutin were used as standards in the following concentrations 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.7812, 0.391 and 0.195 μM. Blank solution was prepared by mixing 2 mL of methanol with 4 mL of 50 μM DPPH solutions. The difference in absorbance between the test and the control (DPPH in methanol) was calculated and expressed as % scavenging of DPPH radical. The capability to scavenge the DPPH radical was calculated by using the following equation:

Finally, the IC50 value, defined as the concentration of the sample leading to 50 % reduction of the initial DPPH concentration, was calculated from the separate linear regression of plots of the mean percentage of the antioxidant activity against concentration of the test extract (μg/mL).

Prostaglandin synthesis (COX) inhibition assay

Anti-inflammatory activity was determined using the COX-1 and COX-2 inhibitory assays as described18,19. Indomethacin, a known COX-1 and COX-2 inhibitor was used as control. A stock solution of COX-1 enzyme (60 ) stored at -70 °C was activated with 1250  of co-factor solution (0.3 mg/ml Epinephrine, 0.3 mg/ml reduced glutathione in 10 ml of Tris buffer pH 8.0, and 100 L hematin solution) and pre-incubated on ice for 5 mins. Samples were first screened at 10 mg/mL (crude extracts) and 1 mg/ml (compounds). In duplicate, plant extract (2.5 ) was added to 17.5 of distilled filtered water in Eppendorf tubes to give a final concentration of 250 /mL of organic extract per test solution. A similar preparation of 5 M indomethacin was used as a positive control. Solvent blank and background (2.5 ethanol + 17.5  of water) were used as negative controls. The enzyme solution (60 ) was added to the test solution and pre-incubated at room temperature for 5 mins. The reaction was started by adding 20  of 14C-arachidonic acid to the test solutions, with the enzymes in the background being inactivated by adding 10  2N hydrochloric acid (HCl) before incubating the test solution at 37 °C for 10 mins.

The incubation was stopped and the reaction terminated by adding 10  2N HCl to each test solution. Unlabeled prostaglandins (4  of 0.2 mg/mL) as a carrier solution were then added to each test solution. The samples were loaded onto individual Pasteur pipettes packed with silica gel (particle size 0.063-0.200 mm, Merck) with 1 mL of hexane: 1, 4- dioxan: acetic acid (70:30:0.2 v/v/v) to separate prostaglandins from the unmetabolized arachidonic acid.

Column chromatography was used to elute the unmetabolized arachidonic acid with 4 mL of hexane: l, 4-dioxan:acetic acid (1 mL at a time) while the elution and collection of prostaglandin products was done with 3 mL of ethyl acetate: methanol (85:15 v/v) into scintillation vials. Four mL of scintillation fluid was added to the eluate in the scintillation vials and the radioactivity was measured using a Beckman LS 6000LL scintillation counter. Percentage inhibition of the screened extracts was calculated using:

   

DPM = Disintegrations per minute

Results

Table 1: Result of the phytochemical analysis of the methanol extract of Cochlospermum planchonii leaf

Test Reagent           

Observation    

Inference

Unsaturated Steroids/triterpenes (Liebermann-Buchard’s test)

Upper layer: blue-green colour, lower layer: reddish colour

Present

Cardiac glycosides (Keller-Kiliani test)

Pale green colour at the interface (deoxy sugar)

Present

Saponins (Frothing/ Heamolysis Test)

Honey comb froth/heamolysis of RBCs

Present

Flavonoids (Shinoda Test)

Yellow colour

Present

Tannins (FeCl3)

Greenish-Black ppt.

Present

Alkaloids (Dragendorff’s and Mayer’s Test)

1. Cream ppt

2. Reddish brown ppt

Present

ppt = precipitate, RBC = red blood cells

 

Table 2: Result of the thin layer chromatography of the methanol extract of Cochlospermum planchonii leaf

Spot Position (cm)

Rf-value (cm)

Daylight

UV-254 (nm)

UV-366 (nm)

Anisaldehyde spray

0.9

0.13

Yellow

Yellow

Greenish

Greenish

1.7

0.24

Yellow

Yellow

Greenish

Greenish

2.8

0.40

Light Green

Light Green

Greenish Blue

Greenish Blue

4.2

0.60

Brown

Brown

Brown

Brown

5.1

0.72

Yellow

Yellow

Brown

Brown

5.4

0.77

Light Green

Light Green

Greenish Blue

Greenish Blue

6.1

0.84

Brown

Brown

Greenish Blue

Greenish Blue

 

Table 3: Result of the effect of the methanol extract of Cochlospermum planchonii leaf on prostaglandin synthesis

Sample

Prostaglandin Synthesis (%)

COX-1

COX-2

Methanol extract

45.4 ± 6.2

-

Indomethacin

46.3 ± 3.2

-

 

Discussion

Inflammation is a complex immune response in living organisms that helps to defend them against harmful stimuli such as mechanical injury, pathogens, or irritants20. Initiation of the inflammatory process leads to release of a number of mediators and persistence of low-grade inflammation has been linked to the pathogenesis of various disorders, including cancer, atherosclerosis, rheumatoid arthritis, type-2 diabetes, and vascular diseases20,21. Quite a number of steroidal and non-steroidal anti-inflammatory drugs (NSAID’s) are presently being used in the treatment of inflammatory disorders. They are however associated with numerous side effects such as cardiovascular, renal failure and gastrointestinal bleeding22. Researchers are therefore putting a lot of effort into the development of new anti-inflammatory agents from natural sources that will hopefully have fewer adverse effects than the synthetic drugs23-25. In this study, the methanol leaf extract of Cochlospermum planchonii was evaluated on its ability to inhibit COX-1 and COX-2 enzymes which are involved in the biosynthesis of prostaglandins. The COX assay (Table 3) was carried out with the view to ascertaining the mechanism by which the methanol extract exhibits its anti-inflammatory action. The extract was found to inhibit COX-2 selectively at (45.4 ± 6.2%). This is hugely beneficial as COX-2 selective inhibitors have the advantage of lower risk of gastrointestinal bleeding when compared to non-selective inhibitors. The result is also in agreement with a previous study26 which used the Carregenan induced paw edema method in rats to establish the anti-inflammatory activity of the methanolic root extract of the plant.

The phytochemical constituents of the methanolic leaf extract were investigated and it revealed the presence of phytochemicals such as steroids/terpenes, cardiac glycosides, saponins, tannins, flavonoids and alkaloids (Table 1). An earlier report had also found that the methanolic root extract of the plant contained similar phytochemicals such as flavonoids, glycosides and tannins26. Some of these classes of phytochemicals have been linked with a number of activities. For example, Saponnin-rich plants have been shown to demonstrate anti-inflammatory and anticancer properties27 while polyphenolics have been shown to have antioxidant activity among others28. The chromatogram of the extract showed abundant greenish spots when viewed under UV lights of 254 and 366 nm. This can be attributed to the presence of phenolic compounds29. Furthermore, Flavonoids are known to have potent anti-oxidant and anti-inflammatory properties. They have the ability to scavenge free radicals such as hydroxyl and lipid peroxy radicals30,31 which have been implicated in causing some diseases such as diabetes, Parkinson and Alzheimer’s diseases. The presence of these phytochemicals in the extract may therefore account for its traditional use in treatment of some diseases.

Fig 1. Free radical scavenging activity of Rutin & Gallic acid

Fig 2. Free radical scavenging activity of Vitamin C

Fig 3. Free radical scavenging activity of methanol leaf extract

The antioxidant activity of the extract was determined using the 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay and employing ascorbic acid as standard control (Fig 2). The extract had significant antioxidant activity of 93 ± 0.11% in the DPPH assay as compared with ascorbic acid, rutin and gallic acid which had antioxidant activity of 73 ± 0.12%, 90 ± 0.13%., 73 ± 0.15 % respectively. The preliminary antioxidant screening indicated that the extract possesses the potential to scavenge free radicals and the capacity to bleach DPPH even better than all the controls. This can, as well, be attributed to the presence of phenolic compounds31.  

Conclusion

The methanol leaf extract of Cochlospermum planchonii has a selective COX-2 inhibitory activity against prostaglandin synthesis due to the presence of phenolic compounds which are also responsible for the antioxidant activity. Additional work is currently on going in our laboratory in an effort to isolate, characterize and identify these specific compounds for further drug development.

Acknowledgements

The authors are grateful to the Department of Pharmacognosy and Traditional Medicine, University of Jos, Nigeria for providing bench space for the research work.

Source of funding

This research was supported financially by the African Center of Excellence in Phytomedicine Research and Development (ACEPRD), University of Jos, Nigeria.

Conflict of interest

The authors declare that they have no conflict of interest.

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