Research Article

Evaluation of pharmacological potentials of Jatropha curcas Linn sap Euphobiaceae family

S. Abubakar1*, B. O. Akanbi2, C. Osuji1, O. O. Olajide3, E. A. Phillip2

1Sheda Science and Technology Complex (SHESTCO), Biotechnology Advance Research Centre, P.M.B. 186, Garki Abuja, Nigeria

2Department of Microbiology University of Abuja. P.M.B. 117, F.C.T., Abuja, Nigeria

3Chemistry Advance Research Centre, P.M.B. 186, Garki Abuja, Nigeria

*For correspondence

Dr. S. Abubakar,

Sheda Science and Technology Complex (SHESTCO), Biotechnology Advance Research Centre, P.M.B. 186, Garki Abuja, Nigeria.

Email: sal4research@gmail.co m

 

 

 

 

 

 

 

 

 

 

 

 

Received: 22 March 2016

Accepted: 30 April 2016

ABSTRACT

Objective: The study focused on qualitative screening of phytochemical constituents of Jatropha curcas crude sap and its effectiveness on some pathogenic bacteria namely, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Escherichia coli. The anti-oxidant potentiality of the crude sap was also evaluated.

Methods: The sap of J. curcas was collected directly into sterile microfuge tubes after detaching the leaf stalks from the stem, the crude sap (sample) was taken to the laboratory and kept refrigerated at 40C. The crude sap was subjected to preliminary phytochemical screening using standard procedures. The susceptibility test of the sap was carried out using well diffusion method. The anti-oxidant potentiality of the sap was also evaluated using 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) assay.

Results: Based on qualitative phytochemical screening, alkaloids, tannins, saponins, terpenoids, glycosides, and steroid were confirmed present while flavonoids, saponins, balsams were absent. The sap showed appreciable activities against the tested strains of bacteria. The highest zone of inhibition was shown on S. typhi (17 mm) at the crude sap and the lowest zone of inhibition was on P. aeruginosa and E. coli (11 mm) at 1:2 dilution. The antioxidant potential of the crude sap evaluated by UV-Visible spectrophotometer at 517 nm using inhibition of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) is less potent in comparison with Vitamin C, however, it showed increased in activity with decreased concentration.

Conclusions: The J. curcas sap is very effective against all the organisms. Despite the low antioxidant property of the sap, J. curcas sap can be said to have a broad spectrum action. Based on this preliminary work the use of J. curcas to treat diseases caused by microorganisms still remains promising.

Keywords: Pharmacological potential, Jatropha curcas, Phytochemical screening, Antimicrobial activity, Antioxidant activity

Introduction

Plants are the most important sources of medicine. Plant derived compounds (phytochemical) have been attracting much interests as natural alternatives to synthetic compounds.1 Extracts of plants were used for the treatment of various diseases and this formed the basis for all traditional systems of medicine.1 The treatment and control of diseases by the use of available medicinal plants in a locality will continue to play significant roles in medical health care implementation in the developing countries.2 Jatropha curcas is belong to Euphorbiaceae family, a perennial, monoecious shrub or small tree up to 6 m high.3 The plant has thick glabrous branches, straight trunk and grey or reddish bark, masked by large white patches. It has green leaves with short shallow lobs, which are alternately arranged.4 J. curcas is widely used in traditional medicine in Africa, Asia and Latin America to cure various ailments such as skin infections, diarrhea, gonorrhea, fever and several other diseases caused by microorganisms.5,6 J. curcas has also been used as antidote, remedy, medicine and potential source of herbal drugs in dental complaints and against constipation.7

Sap (latex) is applied directly to wounds and cuts as a styptic and astringent to clean teeth, gums, and to treat sores on the tongue and in the mouth, it has coagulating effects on blood plasma.3 The sap also used as a remedy for alopecia, anasorca, burns, dropsy, eczema, inflammation, paralysis and yellow fever.8 The sap is also used for the treatment of dermatomucosal diseases.8

However, there is insufficient information regarding the antimicrobial activities of J. curcas (sap). Whatever limited information available on the medicinal properties of J. curcas is mostly on the leaf extracts of the plant. Therefore, the focus of this work is to evaluate the antibacterial as well as antioxidant property of J. curcas crude sap.

Materials and Methods

Study area

The study area for this research work is Federal Capital Territory (FCT) Abuja, Nigeria. The FCT lies between Latitude 8.25 and 9.20 North of the Equator and Longitude 6.45 and 7.23 East of the Greenwich Meridian, Abuja is geographically located in the centre of the country. The FCT has a landmass of approximately 7.315 km2 of which the actual city occupies 275.3/sq km, it is situated within the Savannah region with moderate climatic conditions.

Figure 1: J. curcas tree and crude sap.

Plant sap collection and preparations

The plant fresh sap from J. curcas was collected directly into microfuge tubes after leaf and stem cutting and kept refrigerated at 4°C. The samples were divided into two parts prior to the analysis. One part was dissolved in 1% DMSO and used for the antimicrobial analysis while the other part was dried using water and used bath at 600C for antioxidant assay.

Sterilizations

This was carried out using autoclave which was done at a temperature of 1210C and pressure of 15 psi for fifteen minutes. It was used to sterilize media, glass wares before use. Alcohol was used to clean the benches and glass wares. Sterilization was done by exposing Jatropha curcas sap to Ultra-Violet rays for 48 hours and afterwards the sap were filter-sterilized using a membrane filter (pore size 0.45 µm) (Sterlitech). Which was carried out by placing the membrane filter on a syntad funnel fixed to a sterile buchner flask connected to a vacumm pump which sucked out the sap.

Collection of test organisms

The test organisms were clinical isolates obtained from the diagnostic laboratory of University of Abuja Teaching Hospital, Gwagwalada, Abuja, Nigeria. They are: Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumonia and Escherichia coli.

Phytochemical screening of the J. curcas crude sap

The J. curcas crude sap was qualitatively analysed for the presence of alkaloids, flavonoids, tannins, saponins, glycosides, terpenoids, phlobatannins, reducing sugars, anthraquinones and volatile oils as follows:

Phytochemical screening

The extracts were qualitatively screened for phytochemicals in accordance with Trease and Evans.9,10

Test for tannins

0.5 g of dried crude sap was stirred with about 10mls of distilled water and then filtered. Few drops of 1% ferric chloride solution were added to 2 ml of the filtrate. The occurrence of a blue –black, green or blue green precipitate indicates the presence of tannins.

Or a dirty green precipitate on addition of few drops of 5% FeCl3 to each of the test sap extracts.

Test for steroids and triterpenoids

Salkowski test: Crude sap extract was mixed with chloroform and a few drops of conc. H2SO4 were added. After vigorous shaking and allowed to stand for some time, a red colour appeared at the lower layer which indicated the presence of steroids and formation of a yellow coloured layer indicated the presence of triterpenoids.

Test for glycosides

5 ml H2SO4 was added to each of the test crude sap in a separate test tube. The mixture was heated in boiling water for 15 min. Fehling solutions A and B were then added and the resulting mixture was heated to boil. A brick red precipitate was obtained indicating the presence of glycosides.

Test for anthraquinones

Borntrager's test: About 0.2 g of dried crude sap was shaken with 10ml of benzene and then filtered followed by addition of 0.5 ml of 1% ammonia solution and shaken. Appearance of a pink, red or violet colour in the ammonical (lower phase) was taken as the presence of free anthraquinones.

Test for saponins

1 g of dried crude sap was boiled with 5ml of distilled water and filtered. To the filtrate, about 3ml of distilled water was further added and shaken vigorously for about 5min.Frothing which persisted on warming was taken as the presence of saponins.

Test for phenols

Half a gram (0.5g) of the dried crude sap was added to 1% ferric (III) chloride in methanol /water (1:1).A dirty green precipitate indicates the presence of phenols.

Or the deep bluish green precipitate formed when equal volume of the sap (extract) was mixed with equal volume of FeCl3 solution. .

Test for alkaloids

Half a gram (0.5 g) of dried crude sap was stirred with 5 ml of 1% aqueous HCl on water bath and then filtered.1ml of the filtrate was taken individually into 3 test tubes. The formation of whitish precipitates when 1 ml of Mayer's reagents, was added indicates the presence of alkaloids.

Test for cardenolides

Two millilitres (2 ml) of benzene was added to 1ml of crude sap sample. The formation of a turbid brown colour is an indication of the presence of cardenolides.

Test for terpenoids

Half a millilitre (0.5 ml) of acetic anhydride was mixed with 1 ml of crude sap sample and a few drops of conc. H2SO4 were added. A bluish green precipitate indicates the presence of terpenoids.

Test for carbohydrate

Fehling's Test for Reducing Sugar: Five millilitres (5 ml) of mixture of equal volume of Fehling's solution A and B was added to 2 ml of crude sap sample in a test tube. The resultant mixture was boiled for 2 mins. A brick red precipitate of copper (i) oxide indicate positive test.

Test for flavonoids

1 g of the crude sap was dissolved separately in dilute NaOH. A yellow solution that turned colourless on addition of conc. HCl indicates the presence of flavonoids.

Test for pholobatannins

A few drops of 1% HCl were added to 1ml of crude sap sample and boiled. A reddish precipitation indicates the presence of phlobatannins.

Test for resins

Two millilitres (2 ml) of crude sap sample plus 2 ml of acetic anhydride solution, by additional drops of conc. H2SO4 gives colophony resins (violet colouration indicates the presence of resins).

Test for Balsams

Three (3) drops of alcoholic FeCl3 is added to 4 ml of crude sap sample which is then warmed. Presence of a dark green colouration is an indication of balsams.

Test for volatile oil

A small quantity of the sample was shaken with dilute NaOH and 0.1 M HCl. A white precipitate was formed with volatile oils.

Confirmation of the test organisms

The identity and purity of the test organisms were confirmed as described by,11-13as follows:

Staphylococcus aureus

An inoculum of the test organism was cultured on mannitol salt agar. The growth produced yellowish colonies. This is due to their ability to use mannitol as food source leading to the production of acidic by-products of fermentation that will lower the pH of the medium and caused the pH indicator phenol red to turn yellow. The colonies were further subjected to Gram staining. The test organism was inoculated on a nutrient agar slant in a bijoux bottle and refrigerated at 4oC.

Escherichia coli

The isolate obtained was cultured on Eosin methylene blue agar by streaking from 18-24 hours colonies with distinctive metallic green sheen were observed which indicate a positive result for E. coli. The colonies were further subjected to Gram staining. The test organism was inoculated on a nutrient agar slant in a bijoux bottle and refrigerated at 4oC.

Pseudomonas aeruginosa

The isolate obtained was cultured on nutrient agar and streaked evenly and left for 18-24 hours. Colonies of fluorescence green colour were observed with round shape and rough surface.

Salmonella typhi

The colony was subjected to Salmonella Shigella agar and the production of translucent colorless colonies on SSA appear with black centre, subcultured on Deocycholate Citrate Agar (DCA). A hydrogen sulphide production by Salmonella spp. It was further cultured on Simmon's Citrate Agar slant for 24 hours. A deep blue colour was observed which indicated a positive result.

Klebsiella pneumonia

Klebsiella species exhibit mucoid growth, large polysaccharide capsules, and lack of motility, and they usually grow positive tests for lysine decarboxylase and citrate, and ornithine decarboxylase and produce gas from glucose.

Antibacterial activity testing

The agar well diffusion method as described by Okoli and Iroegbu,14,15 was employed to determine the growth inhibition abilities of the test organisms by the crude sap. Mueller - Hinton agar was prepared and 30ml each was poured into sterile petri dishes. The agar was allowed to solidify and dry. The agar was aseptically inoculated uniformly with the test organism by flooding with 0.2 ml suspension of (106 cfu) which equivalent (0.5 McFarland) standard the test culture was preserved undisturbed for 30 minutes. With the aid of a sterile 6mm diameter cork-borer, four wells were borne on the agar sufficiently separated and kept at least 15 mm from the edge of the plate and 25 mm from well to well to prevent overlapping of zones.

With the aid of a micropipette 0.2 ml of a known concentration of the crude sap at different concentrations were introduced into separate labelled wells (holes). This was done in duplicates and inoculated plates were incubated at 37 ºC for 24 hours. With the aid of a meter rule, the zone diameters of inhibition were measured and recorded to the nearest millimetre.14 Chloramphenicol was prepared at 10 µg/ml to serve as the positive control.

Minimum inhibitory concentration (MIC)

The MIC of isolates was carried out using tube dilution technique as described by Doughari et al.16 The McFarland standard (106 cfu/ml) was used to standardize the concentration of test organisms. A tube containing 2 ml of 18 hrs nutrient broth without extract was seeded with a loopful of the test organism previously diluted to 0.5 McFarland standard to serve as the positive control while a tube containing 2 ml of 18 hrs nutrient broth that was not inoculated served as the negative control. After incubation for 24 hours at 37oC, the tubes were then examined for microbial growth (turbidity) spectro-photometrically.

Minimum bactericidal concentration (MBC)

The MBC is the lowest concentration of antibacterial substance required to produce a sterile culture. In this technique, the content of the test tube resulting from MIC was streaked using a sterile wire loop on agar plate and incubated at 370C for 24 hours. The lowest concentration of the extract which showed no bacterial growth was noted and recorded as MBC.17

Anti-oxidant activities of J. curcas sap

Scavenging effects on 2, 2-diphenyl-1-picryl hydroxyl radical (DPPH). The radical scavenging activities of the crude sap against DPPH (sigma-Aldrich) were determined by UV-visible Spectrophotometer at 517 nm. Radical scavenging activity was measured by a slightly modified method previously described. 18,19 The following concentration of sap were prepared, 5, 2, 1, 0.1, 0.5 and 0.05 mg/ml in methanol (Analar grade). Vitamin C (Ascorbic acid) was used as the antioxidant standard concentration of 5, 2, 1, 0.1, 0.5 and 0.05 mg/ml. 1 ml of crude sap was placed in test tube and 3 ml of methanol was added, following by 0.5 ml of 1mM DPPH in methanol and thereafter the decrease in absorption was measured on a UV-Visible Spectrophotometer 10 min later. A blank/control solution was prepared containing the same amount of methanol and DPPH. The actual decrease in absorption was measured against that of the control and the percentage inhibition was calculated. All test and analysis were run in duplicates and the results obtained were averaged. The radical scavenging activity (RSA) was calculated as the percentage inhibition of DPPH discolouration using the equation below:

% inhibition =

Where, Ab is the absorption of the blank sample (without the sap) and Aa is the absorption of the sap.

Statistical analysis

An IBM SPSS 20 standard deviation was used to find whether there is variation in the activities of the crude sap on tested organisms (p≤0.05).

Results and Discussion

Table 1 below shows the physical characteristics of the J. curcas fresh crude sap that was used to carry out the research work.

Table 1: Physical characteristics of J. curcas sap.

S. No.

J. curcas

Sap

1

Colour

Brownish

2

Odour

unpleasant

3

Texture

oily

Table 2: Phytochemical constituents of the J. curcas sap.

S/No.

Bioactive compounds

Indicators

1

Alkaloids

+

2

Flavonoids

-

3

Terpenoids

+

4

Anthraquinones

-

5

Saponins

+

6

Tannins

+

7

Phlobatanin

-

8

Glycosides

+

9

Volatile oil

-

10

Steroids

+

11

Cardinolides

-

12

Resins

-

13

Balsams

-

Keys: + = Present and - = Absent

The qualitative phytochemical analysis carried out on the crude sap of J. curcas showed the presence of tannins, alkaloids, terpenoids, glycosides, steroid, while flavonoids, anthraquinones, saponins, phlobatannin, volatile oil, cardinolides, resins, balsams were absent. These compounds are all known to be biologically active therefore they aid in the antimicrobial activities. These secondary metabolites exert antimicrobial activity through different mechanisms. The Crude sap contain very prominent and medicinal compounds such us alkaloid which is used as an antimalarial, analgesics, and stimulants.20 Steroids have been reported to have antibacterial properties,21 and they are very important compounds especially due to their relationship with compounds such as sex hormones.22 Alkaloids have been associated with medicinal uses for centuries and one of their common biological properties is their cytotoxicity.23 Several workers have reported the analgesic,24 antispasmodic and antibacterial,25,26 properties of alkaloids. Glycosides are known to lower the blood pressure according to many reports.27 Tannins are polymeric phenolic substances found in nearly all plants part.28 Tannins have been known to display different biological activities including antifungal and antibacterial.29 Some of these bioactive compounds that are synthesized as secondary metabolites as the plant grows are also used to protect the plant against microbial attacks and predation by animals.30 The crude sap were also revealed to contain saponins which are known to produce inhibitory effect on inflammation.31 Saponins has the property of precipitating and coagulating red blood cells. Some of the characteristics of saponins include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding properties and bitterness.32,33

Table 3: Zone Diameter of inhibition (mm) of the J. curcas sap test of organisms and both positive and negative control.

Microorganisms

Zone diameter of inhibition in (mm) and Concentration (mg/ml)

Crude sap

1.1

1.2

+ve

-ve

Klebsiella pneumonia

16

13

12

37

-

Pseudomonas aeruginosa

16

14

11

35

-

Escherichia coli

14

13

11

38

-

Staphylococcus aureus

15

13

12

33

-

Salmonella typhi

17

15

13

38

-

Keys: +ve = positive; -ve = negative '-' = no activity

The antimicrobial investigation carried out on the J. curcas sap showed a broad spectrum of activity as presented in Table 3. The sap showed appreciable activity against the different strains of pathogenic bacteria used. The activity of the extract showed concentration dependent with the highest zone of inhibition pronounced on crude extract. The highest zone was showed on S. typhi (17 mm) while K. pneumonia and P. aeruginosa showed (16 mm) zone of inhibition each respectively. The sap showed a low level of effectiveness compared to the commercial antibiotic (chloramphenicol) which was used as positive control.

Table 4: MIC and MBC of J. curcas sap.

Organisms

1:1

1:2

0.5:2.5

0.25:2.85

K.

pneumonia

0.334**

0.671*

0.977

1.388

P.

Aeruginosa

0.479**

0.790*

0.957

1.262

S. typhi

0.427**

0.581*

0.648

1.497

S. aureus

0.277

0.133**

0.580*

0.686

E. coli

0.666**

0.757*

0.984

1.087

Key: Values with * represent MIC. While values with** represent MBC.

The turbidity spectrophotometric reading for the MIC of the J. curcas sap in this study showed that with exception of S. aureus all the test isolates showed MIC at the concentration (1:2 v/v), the K. pneumonia, P. aeruginosa, S. typhi, and E. coli showed MBC at (1:1) values recorded 0.334, 0.479, 0.427 and 0.666 respectively.

The S. aureus showed MBC at (1:2 v/v) valued recorded 0.133 spectrophotometrically, as showed in Table 4.

Figure 2: Antioxidant activity curve of J. curcas sap against vitamin C (standard).

J.C.S=J. curcas sap, Vit C=vitamin C (standard)

Many researchers have been searching for powerful of non-toxic antioxidants from natural sources, especially edible or medicinal plants. Natural antioxidants have been studied in order to find out compounds protecting against a number of diseases related to oxidative stress and free radical-induced damage34. Results have shown that the raw extracts or isolated pure compounds from them were more effective antioxidants in vitro than BHT or vitamin E So, medicinal plants can be a potential source of natural antioxidants.35

A rapid simple and inexpensive method to measure antioxidant capacity of food involves the use of the free radical, 2, 2,-diphenyl-1-picrylhydroxyl (DPPH) which is widely used to test the ability of the free radical scavengers or hydrogen donors and to evaluate antioxidant activity. When antioxidants react with DPPH, it becomes paired off in the presence of a hydrogen donor and is reduced to DPPH-H form, results in decolourization with respect to the number of electrons captured. The more the decolourization the more is the reducing ability. The sap exhibited scavenging ability at lower concentration compared with the standard (Vitamin C). Lower absorbance of the reaction mixture indicated higher free radical scavenging (antioxidant) power of the sap. This may be as a result of absence of flavonoids which is an important secondary metabolites and it also aids scavenging activity.

The antioxidant feature of the crude sap evaluated by UV-Visible spectrophotometer at 517nm using inhibition of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) is less potent in comparison with Vitamin C, figure 2. However, it showed increased in activity with decreased concentration. The IC50 value recorded from the graph is 2.289. IC50 value is defined as the amount of antioxidant agents required to decrease the initial DPPH concentration by 50%.

Conclusions

From the study Jatropha curcas sap is very effective against all the tests organisms. The presence of tannins, saponins, alkaloids and steroids in J. curcas crude sap supports the traditional medicinal uses of this plant in the treatment of different ailments.

Funding: No funding sources

Conflict of interest: None declared

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