Volume 3 Issue 2 October 2015 ISSN: 2347-1697 ... Ficus Indicia, Antimicrobial Activity, Anthelmintic Activity, Phytochemical Screening, Physicochemical Property, Fluconazole, Tetracycline,

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Available online through - http://ijifr.com/searchjournal.aspx Accepted After Review On: October 17, 2015

Published Online On: October 21, 2015

International Journal of Informative & Futuristic Research

ISSN: 2347-1697 Volume 3 Issue 2 October 2015

Physicochemical Properties, Phyto-Chemical

Screening, Antimicrobial Activities, Anti-

Helmantic Activities And Nutritional Values Of

Cactus (Opuntia Ficus Indicia) Around Adigrat Paper ID IJIFR/ V3/ E2/ 025 Page No. 420-435 Research Area Bio-Chemistry

Index Terms

Opuntia Ficus Indicia, Antimicrobial Activity, Anthelmintic Activity,

Phytochemical Screening, Physicochemical Property, Fluconazole, Tetracycline,

Piperazine Citrate

1st Kassa Belay

PhD student

National Taiwan University of science and Technology,

Graduate Institute Of Applied Science And Technology

Assistant Professor,

Department of Chemistry,

College of Natural and Computational Science,

Adigrat University, Adigrat, Tigray- Ethiopia

2nd

Zebib Abisa


Department of Nutrition,

College of Agriculture and environmental science,

Adigrat University

3rd

Taame Abrha


Department of Biology,

College of Natural and computational science, Adigrat

University

4th

Wendie Mebrat




University

5th

Shilash Bedassa




University

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ISSN: 2347-1697 International Journal of Informative & Futuristic Research (IJIFR)

Volume - 3, Issue -2, October 2015 Continuous 26th Edition, Page No.:420-435

Kassa Belay ,Zebib Abisa, Taame Abrha, Wendie Mebrat, Shilash Bedassa:: Physicochemical Properties, Phyto-Chemical Screening, Antimicrobial Activities, Anti-Helmantic Activities And Nutritional Values Of Cactus (Opuntia Ficus Indicia) Around Adigrat

Abstract

The present study deals with the antimicrobial activity, anthelmintic activity and phytochemical screening of the Opuntia ficus indica, this is commonly available in Adigrat. Therefore it was thought worthwhile to explore this indigenous plant for its activity against different microorganisms. The alcoholic extract exhibited significant anti-bacterial, antifungal activity, comparable to the standard drug tetracycline. The petroleum ether and alcoholic extract were evaluated for Anthelmintic activity on adult earthworms, ‘Pheretima posithuma’. The alcoholic extract produced more significant Anthelmintic activity than petroleum ether extract and the activities are comparable with the reference drug Piperazine citrate. Phytochemical analysis of these plants confirms the presence of various phytochemical like alkaloids, flavonoids, phenols, steroidal terpenes , alkaloids, flavonoids, Saponin, tannins, steroidal terpenes, reducing sugar, xanthoproteins, Phenolic compounds. The cactus was found to contain almost all the phytochemicals except for the absence of anthraquinones. These plants can be a source of useful drugs but further studies are required to isolate the active component from the crude plant extract for proper drug development. Chemical proximate analysis and mineral constituent analysis at different maturation stages were carried out in this investigation. As a result, older prickly pads were found to be an important source of nutritional components such as calcium

1. Introduction

Cactus (Opuntia ficus-indica), commonly known as prickly pear, which belongs to the Cactaceae

family. It is one of the most representative fruits in Mexican culture. It is a fruit, which presents a

thick pericarp with small prickles, enclosing a pulp, which is intermixed with a number of small

seeds [1]. Mexico possesses a vast genetic variability, with a diversity of pulp and pericarp

tonalities (purple, red, yellow, green and white) and also with longer harvesting periods, which

include fruits of early, intermediate, and late maturation [2].

It is widely distributed in Mexico and in all American hemispheres as well as in Africa and in the

Mediterranean basin. Cactus is found wild in arid and semiarid plateau regions. It produces sweet,

nutritionally rich edible fruits; its tender cladodes are used as fresh green vegetable and salad. The

fruit, as well as cactus stem are used to prepare value-added products, such as jam, squash, wine,

pickle, body lotions, shampoo, creams, etc. It also has several medicinal and industrial uses. Its

seeds can be used as flavoring agents [5].

But in our country context cactus pear, or beles (Opuntia ficus-indica) is known in East and

Southern Tigray, Ethiopia, is the most important fresh fruit during the summer season. It is widely

dispersed in Northern Ethiopia and according to official estimates, covers 300,000 hectares in

Tigray. However, it is underrated and sold only by street vendors. Using samples from the four

most important locations in Tigray, a basic horticultural description was completed. The study also

included a preliminary census of vendors. The market is based on two yellow-orange varieties:

'Ger’ao' (sweet) and 'Sulhuna' (smooth) as known in the Alitena-Adigrat area; while at the

Maychew-Mehonie area, the dominant cultivars are 'Kile' and 'Limo' which are also yellow-fleshed.

In both areas, they are offered as a mixture. Fruit quality is poor as a result of the primitive harvest

method, and fruit damage ranged from 61 to 86%. Home consumption was estimated at 50%. Beles

is offered as far as Axum (260 km Northwest of Mekelle) and other medium sized towns and small

villages in the area but not in Addis Ababa, the capital city of Ethiopia. According to our census a

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total daily production of 105.26 ton per day was estimated. The retail price was 1.5 birr per kilo (1

US= 8 birr). During the peak of the season, nearly 54.5% of the 3,342 street vendors are located at

Adigrat which is close to the border with Eitrea.It has been used in traditional folk medicine

because of its role in treating a number of diseases and conditions, including anti-inflammatory

effects hypoglycemic effects inhibition of stomach ulceration neuroprotective effects. Through

antioxidant actions and also used for treating diabetes, burns, bronchial, asthma and indigestion in

many countries over the world. One of the most frequently utilized fruit and vegetable technologies

is juice production. Prickly pear fruit is an important source of sugars, minerals, aminoacids,

phenolic compounds ,betalains and vitamin C . Phenolic compounds are localized in the cellular

vacuoles. They play an important role in the growth and reproduction of plants, and also in

protection against pathogens and predators. Phenolic compounds have anti-inflammatory, anti-

allergenic, anti-inflammatory, anti-atherogenic, and cardioprotective effects [8].

The medicinal value of these plants lies in some chemical substances that produce a definite

physiological action on the human body [10], are termed as phytochemicals. Phytochemicals are

bioactive non-nutrient plant compounds that have protective or disease preventive property. They

confer plants with odour (terpenoids), pigmentation (tannins and quinines), and flavor (capsacin)

and are a part of plant naturally defense system. These bioactive components are said to be

responsible for the antimicrobial effects of plant extracts in vitro. They are grouped as flavonoids,

alkaloids, glycosides, Saponin, tannins, terpenoids, carbohydrates, and sterols. Plants have evolved

a number of inducible defense mechanisms against pathogen attack. Some of the responses are

constitutive and pathogen non-specific, but the majority of them are induced after recognition of the

pathogen. Recognition results in the activation of a verity of defense responses, including rapid

localized cell death [9].

Traditional use of cactus as anti-inflammatory effects, inhibition of stomach ulceration, diabetes,

burns, asthma and indigestion in many countries over the world.[12] to prepare jam, wine, body

lotions, shampoo, creams, etc. However in Ethiopia it’s only uses as nutrition (for man and animals)

and for fence are more pronounced. Worldwide journals regarding cactus and its medicinal uses are

few. Its antimicrobial activity has not yet checked on several photogenes. No anthelmintic

efficacy has been reported. Even essential oils of cactus are almost unknown on the science

cladodes.

2. Objectives Of the Present Research

General objective

The main objective of this work is to investigate and characterize chemical composition,

antimicrobial and anthelmintic activities, nutritive content and physicochemical properties of the

essential oil of Opuntia ficus- indicia.

Specific objective

• To conduct a preliminary phytochemical screening on cladodes sample

• To extract the clodeds and fruit by soxhlet extraction

• To conduct in vitro antimicrobial and anthelimntic test

• To study physico-chemical properties of the essential oil

• To compare and contrast the activities of the crude extract and fractions with the standard

drugs and other studies in the plant

• To determine ascorbic acid content

• To determine iron content and its good physiological effect of the cladodes

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3. Materials & Methods

3.1 Description of study area :

The cactus cladode sample was collected from around Adigrat (Erop , Fatsi and Bizet). These towns

are located in Middle Eastern Tigray, Regional State of Ethiopia. Erop located about 17km far to

the north of Adigrat. Adigrat (14o42’ to 14

o11’N and 37

o34’ to 38

o19’E) is located in Northern part

of Ethiopia, which is about 928 km far from Addis Ababa and about 30 km far from of Mekelle

city, the capital city of Tigray regional state. The study area located in altitude range from 200 -

3000 masl and has a uni-modal rainfall distribution with the highest rain falling from June to early

September. The annual average rainfall ranges from 450mm-600mm and the minimum and

maximum temperature ranges from 6 to 210C. This interesting city is well known in local foods like

‘Tihlo’ and cactus which is harvested once in a year. The largest pharmaceutical manufacturing

factory in Ethiopia, Addis Pharmaceuticals Factory SC, is located in Adigrat. Opened in 1992, the

factory has an annual production capacity of 1.2 billion tables, 19 billion ampoules, 10 million

vials, 500,000 capsules, 4 million ointment tubes and 9.6 million bottles of syrup.

3.2 Chemicals and reagents

n-hexane , Ethanol ,Cyclohexane, Di- ethyl ether ,Petroleum ether , Phenolphthalein solution

,Potassium hydroxide, Hydrochloric acid, Silcagel, Chloroform ,Ferric chloride ,Ammonia solution,

Acetic acid, Mayer’s reagent , Sulphuric acid ,Acid anhydride ,Molash reagent ,Muller Hilton agar

and Mc farland standard

3.3 Materials and apparatus

PH meter, Soxhlet extraction, Uv visible spectrometry, TLC, CC, electronic balance and Abie

refract meter.

3.4 Sample collection and preparation

Opuntia ficus indicia leaf was collected from local area of Adigrat town.

3.5 Extract Preparation

The Opuntia ficus indica plants or prickly pads were collected and washed thoroughly in water,

chopped, air dried for a week at 35-400C and pulverized in electric grinder. 150 gm of the powder

subjected to Soxhlet apparatus using solvents such as petroleum ether and alcohol. The solvent was

then removed under reduced pressure, which obtained a greenish- black colored residue. The yield

was 7.4% and 5.9% respectively. The prepared extracts were used for the antimicrobial and

anthelmintic activity.

3.6 Determination of some selected Physicochemical Properties

The PH, Density of the oil was determined by the methods described by [57]. The Saponification

and acid values were determined using the official methods of Analysis (AOAC, 1990).

I. Determination of specific rotation

The extent of optical activity of the oil was determined by a polar meter (model-D) which measures

the degree of rotation. 20 g of sample in100ml of water and its specific rotation was measured.

Specific rotation pure ethyl acetate solvent and ethyl acetate solution of the oil.

The specific ration [α]T

λ = α/l× ρ,

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Where; λ =wavelength of light used in nm, l = path length in decimeters, ρ =density (g/ml).

II. Determination of saponification value

2.5 g sample was added to 25 ml of 0.1N ethanoic acid and KOH and stir ,3 drop of

Phenolphthalein indicator Titrated with 0.5 M HCl Pink color is disappeared

Saponification value = 56.1xN (Vb - Va)/M

where, N= normality of HCl solution, Vb = volume of HCl solution used in blank, Va = volume of

HCl used in sample, M=mass of the oil used.

III. Determination of refractive index

The test plate was attached to the refracting prism of the refract meter provided with the test plate,

by moistening the test plate with the liquid and pressing it against the refractive prism. The light

was focused on the test plate. The instrument was adjusted until the border line the critical angle

coincides with the cross hairs in the telescop and the reading of the refractive index was taken. The

test plate was removed, cleaned and 2 drops of the essential oil of opuntia ficus indica was placed

on the prism and the prism was clamped together firmly. The light source was fixed so that the light

was reflected through the prisms and instrument was adjusted until the border line between the light

and dark halves of the field of view exactly coincides with the cross hairs of the telescope. The

refractive index was then read.

IV. Determination of acid value

25 ml of ethanol and 3 drop of phenolphthalein were Titrated with 0.1N KOH(end point dark pink

color ) Volume of 0.1N KOH will be noted

Acid value = 56.1×N×V/M

Where N= normality of KOH, M=mass of the oil used V=volume of 0.1N KOH used for titration

V. Solubility: solubility of essential oil of opunicia indica cladodes was checked with four

different solvents, which were water, chloroform, alcohol, and petroleum ether.

VI. Specific Gravity

The specific gravity of essential oil was determined by using the density of the sample to the

density of the water. This step was repeated three times and the average was taken.

VII. Determination of pH : The pH of different formulations in 1% w/v (1g: 100ml) and 10%

w/v (10g: 100ml) of water soluble portions of whole plant powder of opunicia indica, were

determined using standard simple glass electrode pH meter [19].the measurement was repeated

three times and the average was recorded.

VIII. Boiling point: 0.5ml of oil was used to fill a wide capillary tube with an inverse thin capillary

inside. This set up was fixed to the stem of thermometer. The thermometer was then partially

immersed in a glycerol path, at a temperature dependent on the sample of essential oil used. The

continuous and rapid flow of bubbles in the wide capillary was taken as boiling point.

3.7 Determination of some selected Nutritional value Preliminary Phytochemical Screening

The Association of Analytical Chemists’ (AOAC, 1990) methods was used in the determination of

Ash, Moisture content, Protein Content, Fat (Lipid) Content and Carbohydrate content. The

preliminary phytochemical screening of the ethanol and water (hot) extracts of whole plant powder

of Opuntia ficus-indica were carried out using Chemical tests on the aqueous extract and on the

powdered specimens using standard procedures to identify the constituents as described by [5], to

detect the presence of different secondary metabolites (phytochemical constituents) such as

alkaloids, flavonoids, saponins, tannins, steroid glycosides, phenols, coumarins, reducing sugars,

protein, anthraquinones, quinines, Fixed oils and fats [8,14 & 23].

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3.7.1 Determination of Phenolic compounds: 2-3 drops of 1% ferric chloride (FeCl3) solution

were added in to 2 ml portions (1%) of each extract. Phenolic compounds produce a deep violet

colour with ferric ions.

3.7.2 Determination of Tannins Ferric chloride test- A small quantity of the extract was boiled

with water and filtered. Two drops of ferric chloride was added to the filtrate, formation of a blue-

black, or green blackish colour in the presence of ferric chloride precipitate was taken as evidence

for the presence of tannins. Or to 2 ml of test solution Con.HNO3 was added along excess ammonia

.the formation of white precipitate was taken as positive control for Tannins [27].

3.7.3. Determination of Flavonoids: To 1 ml of aqueous extract was added 1 ml of 10% lead

acetate solution. The formation of a yellow precipitate was taken as a positive test for Flavonoids

[1].

3.7.4 Determination Coumarins: form a yellow colour with 1% KOH in absolute ethanol. 1 ml of

portions of 1% solutions of each in test tubes was treated with 3-4 drops of 1% KOH in absolute

ethanol.

3.7.5 Determination of Steroid glycosides: Libermann Burchard’s test- Extract was dissolved in

equal volumes of anhydrous acetic acid and chloroform (CHCl3) and cooled to 0°C. The mixture

was transferred to a dry test tube and concentrated sulfuric acid (H2SO4) was introduced to the

bottom of the tube. Formation of a reddish brown or violet- brown ring at the interface of the two

liquids indicates the presence of steroids.

3.7.6 Determination of Alkaloids Mayer’s Test- 1 ml portions of each extract was acidified with

2-3 drops of 1M Hydrochloric acid and treated with 4-5 drops of Mayer’s regent (Potassium

Mercuric Iodide) Formation of a yellow or white coloured precipitate or turbidity indicates the

presence of alkaloids. Dragendroff‟s Test- Extracts were dissolved individually in dilute

Hydrochloric acid and filtered. Filtrates were treated with Dragendroff‟s reagent (solution of

Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids [3].

3.7.7 Detection of Proteins: Xanthoproteic Test- The extracts were treated with few drops of conc.

Nitric acid. Formation of yellow colour indicates the presence of proteins.

3.7.8 Detection of Quinones: To the test sample, sodium hydroxide is added. Formation of blue,

green, or red colour indicates the presence of quinones.

3.7.9 Detection of Anthraquinones: For examining the anthraquinone derivatives prepare a

specimen in potassium hydroxide solution, anthraquinones give blood red colour.

3.7.10 Determination Saponins: Foam Test- 0.5 g of extract was shaken with 2 ml of water. If

foam produced persists for ten minutes it indicates the presence of saponins. Or In 2 ml of test

solution, 2N HCl was added in small amount and shaken. The aqueous layer was decanted and in

this 2 drops of Mayer’s reagent (potassium mercuric iodide) was added. The intense colour foaming

lather was taken as positive test for Saponin [2].

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3.7.11 Detection of reducing sugar: Fehling’s test- To a test tube 1 ml each a Fehling’s A and B

solutions were added and mixed. To this ~2 ml of plant extract was added and heated on a boiling

water bath for ~10 minutes. Formation of brick red or orange precipitate indicates the presence of

reducing sugar/ carbohydrates.

3.7.12 Detection of Carbohydrates (Molisch’s test)

One drop of concentrated sulphuric acid was added to about 1g of the extract, and then three drops

of 1% α-napthol in 80% ethanol were added to the mixture without mixing to form an upper phase.

Formation of brown or purple ring at the interphase indicated the presence of carbohydrates [4].

Test for terpenoids (Salkowski test): To 0.5 g each of the extract was added 2 ml of chloroform.

Concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown coloration of the

interface indicates the presence of terpenoids.

3.7.13. Detection of Fixed oils and fats Spot test- A drop of concentrated extract was pressed in

between two filter papers and kept undisturbed. Oil stain on the paper indicates the presence of oils

and fats.

4. Experimental Design

4.1 Test Microorganisms For Antimicrobial Assay

Pure culture of bacterial - Serratia marcescens , E. coli (gram negative bacteria), Streptococcus

thermophilus (gram positive bacteria , and Pure culture of fungal strains of Candida albicans,

Fusarium oxysporium, Penicillium sp. and Fluconazole as a standard were purchased from

department of microbiology and veterinary medicine at Mekelle university, Mekelle and stored at -

20ºC. Potato Dextrose Agar (PDA) Media was used as nutritive media during the investigation.

Antimycotic activity of crude plant extract was examined by Disc diffusion method. Plant extract

was examined at three concentrations; they were evaluated in triplicate for each fungus. As positive

control for antimycotic assay Tetracycline and fluconazol were used [5].

4.2 Preparation of inoculums

All the cultures were revived on selective media broth and were given the required incubation

conditions specific of each culture. The gram positive (Streptococcus thermophilus) and gram

negative bacteria (Escherichia coli, Serratia marcescens ) were pre-cultured in nutrient broth

overnight in a rotary shaker at 37°C, centrifuged at 10,000 rpm for 5 min, The fungal inoculums

(Candida albicans, Fusarium oxysporium, Penicillium sp. ) was prepared from Potato dextrose agar

medium. Then these cultures were used for antimicrobial assay [6].

4.3 Procedure of Antimicrobial Assay

4.3.1. Antibacterial activity

For assaying antibacterial activity, the agar well diffusion method was used [7]. About 20 ml of

respective growth media was poured into the Petri plates. Once the agar got solidified, culture of

bacteria was spread after mixing with small amount of GM broth. Four holes were made in the

plates (about 5 mm diameter) using a sterile cork borer and equal volumes of the extracts were

transferred into the holes using pipette. Two Petri dishes containing a particular micro-organism

were used for each concentration of the extract. The plates were allowed to stand for one hour for

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prediffusion of the extract to occur and were incubated at 37 ± 2 oC for 24 hrs. At the end of

incubation the plates were collected and zones of inhibition that developed were measured in mm.

4.3.2 Antifungal Activity

The antifungal activity was tested by disc diffusion method [8]. The potato dextrose agar plates

were inoculated with each fungal culture. The filter paper discs (5 mm diameter) impregnated with

varying concentrations of plant extracts. Water was used to dissolve the extract and completely

dried from discs before application on organism-seeded plates. The activity was determined after 72

h of incubation at 28°C. The diameters of the inhibition zones were measured in mm.

4.4 Study Protocol

Antimicrobial activity was determined by Disc Diffusion method. Muller Hinton was used as

medium for bacterial and fungal strains respectively. [9,10] Positive control experiment was carried

out under the similar condition by using tetracycline and Fluconazole (10µg/ml), as it is the broad

spectrum antibiotic used effectively for bacteria and fungus.

The petridishes with the bacteria and fungal cultures were incubated at 37±20 C for 24 hrs and

27±20 C for 48 hrs respectively. The assessment of anti microbial activity was based on the

measurement of diameter of inhibition zone formed. The experiment was repeated thrice and the

results were taken as mean of three readings. [11,12].

4.4.1 Test microorganisms for anthelmintic activity:

The anthelmintic activity was performed according to the method of [13] on adult earthworm

Pheritima posthuma as it has anatomical and physiological resemblance with the intestinal

roundworm parasites of human beings[14,15]. Four groups of approximately equal sized (6-8 cm)

earthworms consisting of six earthworms in each group were released into 50 ml of desired

formulation. Three groups were prepared as control i.e. distilled water, reference i.e piperazine

citrate (20mg/ml) and third of extracts (20, 40, 60 mg/ml). Observations were made for the time

taken for paralysis was noted when no movement of any sort could be observed except when the

worms Time for death of worms were recorded after ascertaining that worms neither moved when

shaken vigorously nor when dipped in warm water (500C) followed with fading away of their body

colour.

4.4.2 Study Protocol

Four groups of approximately equal size earthworms consisting of six earthworms in each group

were used for the present study.

Group-1 Control (distilled water)

Group-2 Standard (Piperazine citrate- 20mg/ml)

Group-3 Pet. ether extract of different concentration (20mg/ml, 40mg/ml, 60mg/ml)

Group-4 Ether extract of different concentration (20mg/ml, 40mg/ml, 60mg/ml).

Observations were made for the time taken to paralysis and death in individual worms. Paralysis

was said to occur when the worms do not revive even in normal saline. Death was concluded when

the worms lost their motility followed with fading away of their body color [16,17].

4.5 Statistical Analysis

Statistical analysis of the results obtained in each experiment was carried out by use of the

statistical analysis software (SAS) and mean values along with standard deviation were recorded.

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5. Results And Discussion Average Physicochemical properties, Preliminary phytochemical screening for various functional

groups, anthelmintic activity, antibacterial and Antifungal activity of Opuntia ficus-indica course

powder are tabulated as Table No. 1, 2, 3,4&5 respectively.

Table 1: Physicochemical properties for Opuntia ficus-indica @ 25 O

C

Properties Values Review literature

PH 5.7 6.02 56

Acid value 10.542±0.48 9.86±0.039

Density 0.9032g/cm3 0.78 g/cm311

Specific Gravity 0.905±0.001 1.03±0.0024

Refractive index 1.231±0.003 0.87±0.00254

Free fatty Acid (F.F.A.) 0.62% 0.70%23

Saponification Value 29.20 mg/KOH/g 31.10 mg/KOH/g18

Boiling point(OC) 108±0.45 107 ±0.33

53

Optical rotation +9.8±0.001 +7.90±0.00152

Vitamin C 24±0.004 22±0.0015

Table 2: Major and Minor mineral composition, of prickly pads powder

Minerals Amount in mg/l

Current work Literature

Cu 4.925 ±0.45 2.92±0.00157

Zn 21.725±1.5 14.30±0.2743

Pb 0.255±0.03 0.086±0.00426

Cr 0.97±0.02 0.120±0.00638

Ni 6.725±0.38 8.17±1.5344

Co 3.475±0.56 4.72±0.03455

Mn 953.945±10.83 360±0.0253

Fe 211.175±6.82 175±4.2823

Cd 7.415±1.24 2.54±0.0854

Table 3: Phytochemical Screening for Opuntia ficus-indica

Components Aqueous extract Ethanoic extract

Phenolic compound +++ +++

Tannins- Ferric chloride test + +

Flavonoids-shinoda test +++ +++

Coumarins ++ +

Steroid-glycosides-L. Burchard’s test ++ ++

Protein-Xanthoproteic test ++ +

Alkaloid -Mayer’s test

-Dragendroff’s test +++ +++

Quinine + ++

Carbohydrates +++ +++

Anthraquinone - -

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Saponins- foam test +++ +++

Reducing sugars- Fehling’s test ++ ++

Fixed oil and fat-spot test +++ +++ Absence of the phytochemical (-), Trace of the phytochemical (+), Presence of phytochemical (++) and Intense

Presence of phytochemical (+++)

Table 4: Anthelimntic Activity Of Opuntia Ficus-Indica

Group Treatment Con. (Mg/Ml) Time Taken For

Paralysis (Min)

Time Taken For Death

Of Worms (Min)

1st Control(Normal saline) - - -

2nd Piperazine citrate 20 24.66±1.50 55.83±1.45

3rd Petroleum ether Extract 40 90.2±1.7 100±2.6

60 70.2±1.5 90.5±1.5

4th Ethanoic or Alcohol

extract

20 86.2±1.8 90.3±1.0

40 67.8±1.3 80.3±2.0

60 40.0±0.9 50.3±0.8

Figure 1: Anthelimntic activity of Piperazine (Std. Dose) and Extract of opuntia ficus indica with Control

The above figure depicts the Anthelimntic activity of Piperazine citrate (Std. Dose) and Extract of

opuntia indica with Control (Normal saline) against earthworm. The study involves the

determination of paralysis time and death time of the worms in different doses of the extracts (20,

40, and 60 mg/ml). Each Petri-dish contains six number of earthworm. Anthelmintic activity study

of ethanolic extract were performed by comparing with Anthelmintic drug Piperazine as standard

dose. The paralysis time and death time of the worms in different doses of the extracts were noted

shown in table no.4. The control there is no death and paralysis of Indian earthworm. The standard

dose of Piperazine citrate drug at concentration 20 mg/ml show paralysis time and death time at 16

and 23 min. respectively were used as reference. The ethanolic extract of opuntia ficus-indica

leaves concentrations at 60 mg/ml show paralysis time and death time at 18 and 28 min. From the

result it concluded that, ethanolic extract of opuntia ficus-indica leaves were show concentration

dependent anthelmintic activity.

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Table 5: Anti-Bacterial Activity Of Opuntia Ficus-Indica

Treatment Concentration Diameter of Zone of Inhibition (cm)

Bacteria

S. Marcescens E. coli S. thermophilus

opuntia ficus-

indica

Pet. Ether extract

5 mg/ml 1.7 2.0 1.6

10 mg/ml 1.9 2.1 1.8

Alcoholic extract 5 mg/ml 1.9 2.1 1.8

10 mg/ml 2.0 2.4 2.1

Standard Tetracycline 10µg/ml 2.1 2.5 2.3

Table-6: Antifungal activity on different phytopathogenic fungi

Opuntia ficus-

indica

Conc. (mg/ml)

C. albicans F. oxysporium

Penicillium sp.

DIZ (cm)

A.I.

DIZ (cm)

A.I.

DIZ (cm)

A.I.

Cladodes

250 - - - - 0.8±0.15 0.36

500 0.9±0.057 0.45 0.9±0.10 0.47 1.2±0.15 0.54

1000 1.4±0.12 0.7 1.4±0.20 0.73 1.7±0.15 0.77

Control Fluconazole 2.0±0.20 - 1.9±0.20 - 2.2±0.058 -

DIZ= Diameter of inhibition zone; A.I. = Activity index; (-) = No activity Data are presented as mean ±

S.E.M.

6. Discussion

Phytochemical screening of bioactive plants extracts has revealed the presence of alkaloids,

carbohydrates, lactones, proteins, tannins, flavanoids, sterols, terpenes, glycosides, and saponins, of

these, flavonoids and tannins have been linked to antibacterial activity and antidiarrheal activity

[19]. Different phytochemicals display various mechanisms of action such as increasing colonic

water and electrolyte re absorption and inhibiting intestinal motility, while some components have

been shown to inhibit specific pathogens [20]. Phytochemicals such as saponins, terpenoids,

flavonoids, tannins, steroids and alkaloids have anti-inflammatory effects [21]. Steroids and

triterpenoids have analgesic properties [22]. It is therefore probable that these phytochemicals are

responsible for the healing properties of the plants which have been claimed by the farmers in this

study.In this study, tannins were present in the plant tested and may be responsible for the good

antibacterial activity demonstrated by these plant extracts. Previous studies have shown that tannins

have been found to form irreversible complexes with proline-rich proteins resulting in the inhibition

of the cell protein synthesis, they bind proteins and adhesins, inhibit enzymes and complex withcell

wall [23]. Tannic acid which is a mixture of gallic acid esters of glucose can be used as a topical

preparation for cold sores [24]. Many human physiological activities, such as stimulation of

phagocytic cells, host-mediated tumor activity, and a wide range of anti-infective actions, have

been assigned to tannins [25]. Thus, their mode of antimicrobial action may be related to their

ability to inactivate microbial adhesins, enzymes, and cell envelope transport proteins, they also

431




complex with polysaccharide [26]. This probably explains the reason as to why the plant contains

these tannins showed good antibacterial activity.

Basic alkaloids and alkaloid salts were also present in the plant tested and may be responsible for

the good antibacterial activity demonstrated by these plant extracts. Alkaloids have been reported to

be responsible for the antibacterial activity in some plants [27]. Studies have demonstrated that

alkaloids have pharmacological effects and could be associated with inhibition of nucleic acid,

protein, and membrane phosipholipids biosynthesis [30]. The mechanism of action of highly

aromatic planar quaternary alkaloids such as berberine an important representative of the alkaloid

group and humane is attributed to their ability to intercalate with DNA [28]. This probably explains

the reason as to why the plants containing these basic alkaloids and alkaloid salts showed good

antibacterial activity.Furthermore, sterols and sterol glycocides were present in the plant tested and

may be responsible for the good antibacterial activity demonstrated by these plant extracts. Earlier

studies have shown that sterols posses antibacterial and antimicotic activity and have been shown to

act as inhibitors of tumor promotion in vivo [29]. Sterols were found to inhibit tumor promotion in

two-stage carcinogenesis in mice [30]. Sterols were also shown to poses anti-inflammatory activity

after topical application [31]. The presence of these sterols has been reported to account for the

exertion of antimicrobial activity by plants containing them [32]. The presence of these sterols may

contribute to the good antibacterial activity exhibited by these plants.

This sample shows the presence of flavonoids in this study and presence of these may be partly

responsible for the medicinal properties of these plants. Previous studies on other plants have

reported that flavonoids being phenollic compounds are water soluble antioxidants and free radical

scavengers which are capable of preventing oxidative cell damage and have strong anticancer

activity [35]. Reports also say that many disease states are known to be exacerbated by the presence

of free radicals such as superoxide and and hydroxyl and flavonoids have the ability to scacavenge

and effectively mop up these damaging oxidizing species [33]. [34] Reported that the potent

antioxidant activity of flavonoids, their ability to scavange hydroxyl radicals, superoxide anions and

lipid peroxide radicals may be the most important function of flavonoids. The cactus also contained

traces of carotenoids and presence of these may be partly responsible for the medicinal properties of

these plants. The carotenoids are strong antioxidants, being preferentially oxidized over biological

molecules such as nucleic acids and proteins. It is thought that many disease states such as certain

cancers and heart disease are exacerbated by species that cause oxidation; therefore the presence of

these compounds may retard the development of such diseases [36].

7. Findings

7.1 Antimicrobial and anthelimntic screening tests

The antimicrobial and anti-helmintic of the plant extracts varied according to the species of bacteria

tested and the solvents used. From the anthelimntic activity study, the alcoholic extract at a dose of

60mg/ml has significant anthelimntic activity whereas petroleum ether showed moderate activity.

The results of antimicrobial activity of petroleum ether and alcoholic extracts of opuncia ficus

indica were studied and it was found that alcoholic extract of 10mg/ml produced potent

antimicrobial activity as it shows inhibitory zone as compared to other individual concentrations of

432




petroleum ether. The activities are comparable with the reference drug Tetracycline (10µg/ml)

(Table-5)

In conclusion therefore, the study showed that these plants contained compounds of medicinal

importance. It is probable that these constituents were responsible for the antibacterial activity that

they exhibited against the three bacterial species; S. Marcescens, E. coli and S. thermophiles.. These

active compounds in these plants could find place in treatment of various bacterial infections in

poultry where they can be used as alternatives to conventional antibacterial drugs. This could

reduce the unnecessary use of antibiotics in poultry which is making disease-causing bacteria more

resistant to the drugs and diminishing the drugs’ power to treat life threatening disease in humans

and animals.

The results for antimicrobial activity of different plant extracts under study against bacteria are

shown in table-6. The diameter of zone of inhibition decreased with concentration of plant extract.

All the crude seeds extract had shown zone of inhibition against Serratia marcescens, Fusarium

oxysporium and E. coli at all concentrations and zone of inhibition decreased in size (mm) with

concentration of crude seed extract.The crude plant extracts were ineffective to inhibit the activity

of Fusarium oxysporium. This antibacterial potency may be due to the presence of many potent

compounds such as flavonoids, terpenes, phenolics and alkaloids etc .The leaves extract of the plant

species under study were found to contain Tannins, Cardiac glycosides, Terpenoids, Carbohydrates

and Saponin. Phytochemicals are as antimicrobial compounds, have made great contribution for

quick and effective management of plant disease and microbial contamination in several

agricultural conditions.

The leaves extract of the plant species under study were found to contain Tannins, Cardiac

glycosides, Terpenoids, Carbohydrates and Saponin. Cardiac glycosides have anti-inflammatory

activity [37], protect against lethal endotoxemia [38] and are used in cardiac treatment of

congestive heart failure. [39]have reported the membrane disruption and inhibitory effect of

terpenoids against fungi and bacteria. Studies have shown that saponins have heamolytic property,

induced cytotoxicity effect [40], expectorant action [41], antitumor and anti-mutagenic activities

and can lower the risk of human cancers, by preventing cancer cells from growing [42].

Saponins have the property of precipitating and coagulating red blood. These plants are used to stop

bleeding and in treating wounds [44]. They exhibit foaming properties and cell membrane-

permeabilizing properties. Their soapy character is due to their surfactant properties [45]. Thus the

secondary metabolites identified in the plant materials used in the study could be responsible for

antimicrobial activity exhibited by the seeds extracts of the plants. Their varied occurrences in

various plant extracts however indicate that probably, their therapeutic effect(s) are not the direct

effect of a single group or compound, but rather that the compounds possibly act in combination to

bring about an effect.

7.2 Physicochemical Properties

From Table 1, the pH value of sample was determined to be 5.7 this shows the slightly acidic nature

of the oil, it compares favorably with the value of 6.02 reported by [46]. The density of cactus oil

was determined to be 0.9032g/cm3 this is close to the value of 0.9006g/cm3 [47]. The refractive

index was determined to be 1.23; this falls within the range of values reported for some cactus oils,

1.48 for Teleferia occidental seed oil, 1.47 for soybean and 1.47 for corn [48]. The oil yield was

determined to be 27.12%; this is higher than the value of 9.1% reported by [49].

433




The Free fatty acid (FFA) was determined to be 0.62%, this is an important variable in considering

thequality of oil because the lower the FFA, the better the quality of the oil. [50] Reported a value

of 0.37% for cactus oil and added that the lower the FFA, the more its edibility. Therefore, the low

value (0.62%) obtained in this study makes the oil suitable as an edible oil.

The Saponification value indicates that the oil will be good for soap making. The acid value of

cactus oil was determined to be 10.542 mg/KOH/g, the acid value is the mass of potassium

hydroxide (KOH) in milligram that is required to neutralize 1g of the substance, comparing this

value with the acid value of 0live oil of 9.8mg/KOH/g as reported by [51]. Acid value is used to

measure the quality of oil which must not be too high; cactus oil can be oxidized if exposed to light

and comparing it with cocoa butter, coconut oil, palm oil and olive oil, it can be used as lubricant

and plastic

8. Conclusions And Recommendations

8.1 Conclusions

Though there are a number of antibacterial, anti-fungal and anthelmintic drugs available in the

market, they produce many side effects; hence to improve the status of therapy, various

ailments of plant extracts like opuntia ficus indica will be much useful. From the results

obtained, it is clear that if a detailed research is carried out on the alcoholic extract of

opuntia ficus indica, some useful drugs may develop for the treatment of bacterial, fungal

and anthelmintic action.

The study has also demonstrated significant antibacterial and anthelmintic activity

especially in its ether and ethanol extracts. The water extracts have also demonstrated good

antibacterial activity.

This study has further confirmed that this plant contain the phytochemicals which have

healing properties.

The study will solve some of the problems by using cactus to treat a number of bacterial

diseases. The water extracts of these plants demonstrated good antibacterial activity.

Positive assays of tannins, sterols, basic alkaloids and alkaloid salts in this plant clearly

Demonstrate that this plant species may provide bioactive compounds. However in this study I did

not isolate the active compounds responsible for specific antibacterial activity while clinical trials

and toxicity studies were also not carried out. Therefore further research should be carried out on

these and once it is done these will serve as lead compounds in the manufacture of novel drugs

which farmers can use as one of the approaches in prevention and control of diseases. This could

also be an important health and economic resource.

8.2 Recommendations

The extracts of these plants should be further analyzed to isolate the specific antibacterial

Principles in them.

Toxicity studies of the effective plants should also be done to determine the safety indices of

the extracts. Studies to determine the mechanisms of the action, compatibility with other

drugs, side effects and other important parameters should be done.

These plants should be studied more extensively to explore their activity on other organisms

like other bacteria species, protozoa and helminthes among others.

Clinical trials should be carried out to explore the potential of these plant extracts in the

treatment of these infectious diseases.

434




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Documents

Volume 3 Issue 2 October 2015 ISSN: 2347-1697 ... Ficus Indicia, Antimicrobial Activity, Anthelmintic Activity, Phytochemical Screening, Physicochemical Property, Fluconazole, Tetracycline,