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Scheme – I: Successive extraction of fruit peel of Opuntia elatior



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Scheme – I: Successive extraction of fruit peel of Opuntia elatior Mill. 
 
 
93

                                                                                      Materials and Methods 
3.4.2 Qualitative evaluation of peel extracts and OFJ 
The successive peel extracts and OFJ were subjected to various qualitative 
chemical tests to determine the presence of alkaloids, carbohydrate, fats and 
fixed oil, flavonoids, glycosides, phenolics, steroids and terpenoids, wax, 
saponin and betalains phytoconstituents (Kokate, 1996; Evans, 1996; 
Harborne, 2007). 
 
3.4.3 TLC profile of OFJ 
The OFJ was subjected to thin layer chromatography studies using silica gel 
60 F 254 precoated plates (Alugram
®
 SIL G/UV
254
, Macherey – Nagel, 
Germany) to confirm presence of carbohydrates with reference to standard 
using solvent system chloroform:methanol (6:4) (Egon, 2007) and betalains 
using solvent system n-Butanol:Acetic acid:Water (BAW) (4:1:5) and 1 % 
Aqueous Hydrochloric acid (Harbone, 2007). The R
f
 values and color of spot 
were observed and compared. 
 
3.4.4 Qualitative analysis of betalain 
3.4.4.1 Spectrophotometric analysis 
Betalain from the fruits of Opuntia elatior Mill. was extracted using methanol, 
deionized water and methanol containing 1% conc. hydrochloric acid (HCl), 
with a ratio mass fruit (g)/solvent (ml) 1:5. Freshly cut fruit pulp was 
homogenized in these solvents for 1 min and centrifuged at 3000 g at 15 ºC for 
10 min in a Compufuge cooling centrifuge (Remi Instrument, Mumbai). 
Supernatants were filtered through a glass filter G
4
 (Borosil Glass Works Ltd., 
Mumbai), and the filtrate obtained were analyzed using spectrophotometer. 
The spectrum (400 to 650 nm) of methanol, deionized water and methanol 
HCl extracts was recorded on double beam UV-visible spectrophotometer 
(Thermo Spectronic, Cambridge, UK) equipped with VisionPro software V 
4.10, spectral bandwidth of 2.0 nm, wavelength accuracy of ± 0.5 nm and a 
pair of 1 cm matched quartz cells. The identity of absorption spectra was 
confirmed by observed λ
max
 (Farnandez-Lopez & Almela, 2001; Yahia & 
Castellanos-Santiago, 2008). 
 
 
 
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                                                                                      Materials and Methods 
3.4.4.2 High performance liquid chromatographic (HPLC) analysis 
Identification of betalains by HPLC-DAD analysis was performed in a 
Shimadzu HPLC-PDA system comprising of two LC-20 AD liquid pumps, 
automatic rinsing kit for LC-20 AD, Gradient mixer, 7725i manual injector, 
fixed –loop manual kit, 25 µl syringe, injector adapter, SPD-M20A photo 
diode array detector, and LC solution Multi PDA workstation software. 
Analyses were performed using an analytical scale of 25 cm x 4.6 mm i.d. and 
a C18 column with a particle size of 5 µm (Merck), operating at a temperature 
of 25 ºC. The program consisted of a 30 min linear gradient elution from 
solvent A (1% acetic acid in water) to 12 % solvent B (1% acetic acid in 
acetonitrile) with a flow of 1 ml/min. In each analysis, 20 µl of the methanolic 
extract was directly injected onto the chromatographic column. The 
betaxanthin and betacyanin composition of methanolic extract was confirmed 
by their visible spectral characteristics in comparison to literature and 
retention times of chromatographic peaks (Farnandez-Lopez & Almela, 2001). 
  
3.4.4.3 Liquid chromatography – mass spectroscopic (LC-MS) analysis 
HPLC – DAD was coupled with mass spectrometer (TSQ Quantum Ultra, 
Thermo Scientific, USA) equipped with an electrospray ionization source and 
LC-quan software (2.5.6.1) operating in the positive ionization mode. 
Nitrogen gas was used as the dry gas at a flow rate of 12 L/min with 
nebulizing (40 psi). The spectra was take to promote [M+H]
+
 ion production 
(electrospray voltage 4 kV), and nebulizer temperature was set at 200 ºC. The 
program consisted of a 30 min linear gradient elution from solvent A (1% 
acetic acid in water) to 12 % solvent B (1% acetic acid in acetonitrile) with a 
flow of 1 ml/min. The injection volume for fruit extract sample was 20 µl. The 
identification of betanin was confirmed by mass spectrometry (Farnandez-
Lopez & Almela, 2001). 
 
 
 
 
 
 
 
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                                                                                      Materials and Methods 
3.4.5 Quantitative estimation of OFJ 
3.4.5.1 Total sugar content 
The total sugar content was determined by the anthrone reagent method 
(Plummer, 2006). The fruit juice was prepared as described in 3.3.3.2 and 
transferred 1.0 ml of filtered juice to the 10 ml volumetric flask, and diluted to 
10.0 ml with distilled water, having strength 100µl/ml, labelled as stock 
solution. Stock solution (0.1 ml) was transferred into 10 ml volumetric flask, 
and diluted to 10 ml with distilled water, having strength 10µl/ml. The 
anthrone reagent was prepared right before analysis by dissolving 0.2 g of 
anthrone (0.2%) in 100 ml of concentrated sulfuric acid, protected from light 
and used within 12 h. Anthrone reagent (4.0 ml) was added cautiously to each 
tube containing 1.0 ml of standard solutions of glucose (10 – 100 µg/ml) and 
test solution (10µl/ml).  Tubes were then placed 10 min at 5 
0
C. Subsequently, 
tubes were boiled 5 min on constant boiling water bath. After heating, allowed 
to cool at room temperature for 15 min. The absorbance of the colored 
solution was measured at 620 nm against reagent blank. Readings were taken 
in triplicate.  Spectrophotometric response was compared to a standard 
calibration curve of glucose, and total sugar content was expressed as g/100 
ml of glucose. 
 
3.4.5.2 Total phenolic content 
The amount of total phenolics in the extracts was determined according to a 
modification of the Folin-Ciocalteu method (Kujala et al., 2000). A 1.0 ml of 
fruit juice as described in 3.3.3.2 extracted in 10.0 ml methanol:water (50:50 
v/v), having strength 100 µl/ml was introduced into test tube and mixed with 
1.0 ml of 50% Folin-Ciocalteu’s reagent. The mixture was allowed to stand for 
a 2 to 5 min period which was followed by the addition of 2.0 ml of 20% 
Na
2
CO
3
. After 10 min incubation at room temperature, the mixture was 
centrifuged for 8 min (150 g) and the absorbance of the supernatant was 
measured at 750 nm on a UV-visible spectrophotometer against reagent blank. 
Readings were taken in triplicate. Spectrophotometric response was compared 
to a standard calibration curve of gallic acid, and the total phenolic content 
was expressed as gallic acid equivalents in mg/100 ml. 
 
 
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                                                                                      Materials and Methods 
3.4.5.3 Titratable acidity 
The titratable acidity was determined by titration with 0.1N sodium hydroxide 
(NaOH) to pH 8.1 according to AOAC (1995). The glass electrode of 
calibrated pH meter was immersed in 100 ml of fruit juice as described in 
3.3.3.2 in beaker. Initial pH values before titration with 0.1N NaOH was noted 
down. Stirred moderately and added alkali quite rapidly until near pH 6. Then 
add alkali slowly to pH 7. After pH 7 was reached, finish titration by adding 
0.1N NaOH 4 drops at time, and record total volume and pH reading after each 
addition. Continue titration ≥4 drops beyond pH 8.1, and interpolate data for 
titration corresponding to pH 8.1. Readings were taken in triplicate. The 
titratable acidity was expresses in g of anhydrous citric acid/100 ml. 
 
3.4.5.4 Total betacyanin content 
Quantification of total betalain content was carried out according to Cai & 
Corke (1999) and Stintzing et al. (2003) nine times from pigment extracted in 
deionised water as described in 3.3.3.2 without pH adaptation applying the 
molar extinction coefficients of betacyanin (ε =60,000 L/mol cm in H
2
O; 
λ=538 nm; MW=550 g/mol). The pigment extract was diluted with deionized 
water to obtain absorption values of 0.9 ≤ A ≤ 1.1. The total betacyanin 
content was calculated using the equation: Betacyanin Contents [mg/l] = 
[(AxDFxMWx1000)/(εxL)], where A is the absorption at 538 nm for 
betacyanins. DF is the dilution factor and L the pathlength of the 1-cm cuvette. 
For MW and ε, the molecular weights and extinction coefficients of the 
representative compounds betanin have to be considered. 
 
3.4.5.5 Elemental analysis 
The metallic elements were analyzed by atomic absorption spectrometry 
(AAS) using air/acetylene flame (Walsh, 1955). Weigh about 30 ml of fruit 
juice as described in 3.3.3.2 accurately in a silica crucible. Dried and then 
ignited over a bunsen burner till it charred. Transferred to muffle furnace at 
500 ± 50 ºC for ashing till all carbonaceous matter burned and dull grey ash 
resulted. Cool and then treated with 25 ml 6N HCl to dissolve the ash. Heated 
it over steam bath for 15-20 min and transferred the ash solution quantitatively 
to 50 ml volumetric flask and make up the volume. The solution was filtered 
 
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                                                                                      Materials and Methods 
to remove any acid-insoluble matter using Wattsman filter paper no. 14. The 
sample solution obtained was used for further estimation of metal elements 
viz. copper, iron, zinc, calcium, potassium and sodium. The atomic absorption 
spectroscopic analysis was carried out at Consumer Education and Research 
Centre (CERC), Ahmedabad. 
 
Instrumental conditions: 
A double beam Schimadzu (AA-6300) atomic absorption spectrophotometer 
(AAS) was used. 
 
Table 3.1: Instrumental conditions for elemental analysis using AAS. 
Elements 
Instrumental 
conditions 
Copper Iron Zinc Calcium
Potassium Sodium 
Wavelength 
(nm) 
324.8 248.3 
213.9 422.7  766.5 
589.0 
Slit width 
(nm) 
0.7 0.2 
0.7 0.5  1.0 
0.2 
Flame type 
Air – C
2
H
2
Gas flow rate 
(L/min) 
1.6 2.2 
2.0 1.7  1.7 
1.8 
Burner height 
(mm) 
06 09 
07 10  07 
07 
 
 
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                                                                                      Materials and Methods 
3.5 Pharmacological studies 
3.5.1 Plant material 
The fruit juice (OFJ) of Opuntia elatior Mill. was prepared as described in 
3.3.3.2 and used for evaluation of haematinic, analgesic, anti-inflammatory 
and anti-asthmatic actions. 
   
3.5.2 Animals 
Albino wistar rats of either sex (180-250 g body weight) were used for this 
study. They were housed at ambient temperature (22±1
0
C), relative humidity 
(55±5%) and 12h/12h light dark cycle. Animals had free access to Amrut 
brand rat pellet diet supplied by Pranav Agro Industry, Baroda, and water 
given  ad libitum. The protocol of the experiment was approved by the 
Institutional Animal Ethical Committee (IAEC) as per the guidance of the 
Committee for the Purpose of Control and Supervision of Experiments on 
Animals (CPCSEA), Ministry of Social Justice and Empowerment, 
Government of India, vide certificate no. IAEC/RBPMPC/09-10/01 dated 
18/07/2009. 
  
3.5.3 Acute toxicity study 
Acute toxicity studies were performed for fruit juice according to the acute 
toxic classic method as per guidelines 423 prescribed by OECD (2001). 
Female albino rats were used for acute toxicity study. The animals were kept 
fasting for overnight providing only water. Theses were divided into two 
groups of each containing five animals. Each of these groups was then 
administered with water and fruit juice as described in 3.3.3.2 of Opuntia 
elatior Mill. at the dose of 20 ml/kg p.o. The animals were observed for 30 
min and then periodically for first 24 h special attention during first 4 h and 
thereafter daily for 14 days. The observations like sedation, convulsions, 
tremors, lethargy, death etc were systemically recorded with individual 
records of each animal. 
 
 
 
 
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                                                                                      Materials and Methods 
3.5.4 Estimation of haematinic action 
3.5.4.1 Mercuric chloride (HgCl
2
) –induced anaemia 
3.5.4.1.1 Experimental design 
Haematinic action was evaluated by methods previously described by Rathore 
and Siddiqui (2000) and Sarkar et al. (2007) with some modification. Mercuric 
chloride (HgCl
2
) dose was arrived at after carrying out initial pilot studies. The 
dose of OFJ was selected after carry out acute toxicity study. It was found that 
maximum dose (20 ml/kg, p.o.) was safe and based on that we had selected 
three different doses low (5 ml/kg), medium (10 ml/kg) and high (15 ml/kg) 
for this study. Animals were divided in seven groups (n = 6) as per followings 
and treated accordingly. 
 
Group A:  
Negative control (saline solution for 60 days)  
Group B:  
Positive control (HgCl
2
 solution, 4 mg/kg, p.o., upto 30 days) 
Group C:  
HgCl
2
 + Standard ferrous sulphate containing drug Fefol
®
  
(0.0214 mg/kg, p.o., treatment started on day 31 upto day 60) 
(Pandit et al., 1999) 
Group D5:  
HgCl
2
 + OFJ (5 ml/kg, p.o., treatment started on day 31 upto  
day 60)  
Group D10:   HgCl
2
 + OFJ (10 ml/kg, p.o., treatment started on day 31 upto  
day 60)  
Group D15:   HgCl
2
 + OFJ (15 ml/kg, p.o., treatment started on day 31 upto 
day 60)  
Group E:  
OFJ (15 ml/kg, p.o., treatment started on day 31 upto day 60) 
 
Mercuric chloride (4 mg/kg, p.o.) was given to each rat except to group A and 
E for 30 days to induce anemia. After mercuric chloride exposure, treatment 
was given for the next 30 days except in group A and B. Group B animals 
were allowed to recover naturally. Group E animals were treated with only 
fruit juice (15 ml/kg) for next 30 days. Haematological and biochemical 
parameters were estimated on 30
th
 and 60
th
 day. At the end of study, 
histopathological study of liver, kidney and spleen were evaluated. 
 
 
 
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                                                                                      Materials and Methods 
3.5.4.1.2 Measurement of Body weight 
The change of body weight in grams of each animal was recorded at 7-day 
intervals using ACCULAB digital balance, (Model No. ALC-310.3, Sartorius 
Mechatronics India Pvt. Ltd., Bangalore, India). 
3.5.4.1.3 Measurement of Haematological Parameters 
Blood samples were withdrawn from retro-orbital plexus under light ether 
anesthesia, collected in heparinized capillary tubes and analyzed for 
haematological parameters. 
Haemoglobin (Hb) content (gm %) of each animal was estimated by Sahli’s 
haemoglobinometer at 7-day intervals. When blood is added to 0.1 N 
hydrochloric acid, haemoglobin is converted to brown colored acid hematin. 
The resulting color after dilution is compared with standard brown glass 
reference blocks of a Sahli haemoglobinometer. By using a pasteur pipette 
added 0.1 N hydrochloric acid in the tube up to the lowest mark (20% mark). 
Blood was drawn up to 20 µl mark in the Hb-pipette. Blood column adjusted 
carefully without bubbles. Blood was transferred to the acid in the graduated 
tube; reaction mixture mixed and allowed the tube to stand for at least 10 
minutes. The solution was diluted with distilled water by adding few drops at a 
time carefully and by mixing the reaction mixture, until the color matches with 
the glass plate in the comparator. The matching was done only against natural 
light. The level of the fluid was noted at its lower maniscus and the reading 
corresponding to this level on the scale was recorded in gm % of haemoglobin.   
 
Haematological parameters like total red blood cells (RBC), total white blood 
cells (WBC), differential white blood cells, haematocrit, mean cell volume 
(MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration 
(MCHC), platelet count, mean platelet volume (MPV), platelet distribution 
width (PDW) and red blood cell distribution width (RDW) were estimated on 
fully automated fluorescence flow cytometry 5-part different analyzers 
(Sysmex XS800i, Japan). 
 
 
 
 
 
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                                                                                      Materials and Methods 
3.5.4.1.4 Measurement of Biochemical Parameters 
For collection of serum, blood samples were withdrawn from retro-orbital 
plexus under light ether anesthesia without any anticoagulant and allowed for 
10 minutes to clot at room temperature. It was then centrifuged at 2500 g for 
20 minutes. The serum obtained was kept at 4ºC until used. 
 
3.5.4.1.4.1 Estimation of Blood Sugar 
Serum glucose levels were determined by the glucose oxidase – peroxidase 
(GOD-POD) method using Bayer Diagnostics reagent kit (Ahmedabad, India).  
Principal: 
The analysis method is based on a principle that the glucose oxidase enzyme 
when added to serum sample and incubated at 37°C for 15 minutes, converts 
glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide is 
converted to water and oxygen by the enzyme peroxidase. 4-aminophenazone, 
an oxygen acceptor, takes up the oxygen and together with phenol forms a 
pink colored chromogen. The optical density (OD) at 530 nm of the color 
formed is directly proportional to the glucose level (Godkar & Godkar, 2004). 
Procedure: 
Pipette in the tubes labeled as follows; 
 Blank 
Standard 
Sample 
Working Reagent 
2 ml 
2 ml 
2 ml 
Distilled Water 
10 µl - 

Standard - 
10 
µl - 
Sample - 
- 10 
µl 
 
Mix and read the optical density (OD) after 10 minutes incubation. The final 
color is stable for at least 1 hour. 
Calculations: 
Serum Glucose (mg/dl) = Abs. of sample/ Abs. of Std. X 100 
 
 
 
 
 
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                                                                                      Materials and Methods 
3.5.4.1.4.2 Kidney functions study 
Estimation of serum urea and creatinine were carried out by using standard kit 
(Nicholas India Pvt. Ltd., Ahmedabad, India) with semi-auto analyzer 
(photometer 5010).  
3.5.4.1.4.2.1 Estimation of Creatinine (Modified Jaffe Method) 
In vitro quantitative determination of the activity of creatinine in serum was 
done using enzymatic kit (Nicholas India Pvt. Ltd., Ahmedabad, India). 
Principle: 
Creatinine forms a colored complex with picrate in alkaline medium. The rate 
of formation of the complex is measured. 
Procedure: 
 
Prepare a 1:1 mixture of reagent 2 and reagent 3 (Mono reagent) at 
least 10 min. before starting assay. This mixture is stable for 5 days at 
15
0
C- 25
0
C when stored in a dark bottle. 
  
Blank 
Standard 
Test 
Distilled water 
100 µl 


Creatinine standard - 
100 µl 

Test - 

100 
µl 
Monoreagent 
1000 µl 
1000 µl 
1000 µl 
 
Mix and start stopwatch at the same time. After 30 sec. read 
absorbance (A
1
) at 492 nm and exactly after another 120 sec. read 
absorbance (A
2
). 
 
A
sample
 as well as A
std 
 = A
2
 – A

Calculation: Creatinine (mg/dl) = 2  X  A 
sample
 / A 
std 
3.5.4.1.4.2.2 Estimation of Urea (Urease-Glutamate dehydrogenase: 
enzymatic UV test) 
In vitro quantitative determination of the activity of urea in serum was done 
using enzymatic kit (Nicholas India Pvt. Ltd., Ahmedabad, India). 
Procedure: 
 
Mix 4 parts of reagent 1 with 1 part of reagent 2 (Mono reagent). 
Leave the mono reagent for at least 30 min. at 15- 25
0
C before use and 
protected from light. 
 
 
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                                                                                      Materials and Methods 
 
 Blank 
Standard 
Test 
Distilled water  10 µl 


Urea standard 

10 µl 

Test - 

10 
µl 
Monoreagent 
1000 µl 
1000 µl 
1000 µl 
 
Mix and start stopwatch at the same time. After 60 sec. read 
absorbance (A
1
) at 340 nm and exactly after another 60 sec. read 
absorbance (A
2
). A
sample
 as well as A
std 
 = A
2
 – A

Calculation: Urea (mg/dl) = A 
sample
 / A 
std   
X  conc. of Std 
 
3.5.4.1.4.3 Liver functions study 
3.5.4.1.4.3.1 Estimation of Alkaline phosphatase (ALP) 
In vitro quantitative determination of the activity of ALP in serum was done 
using enzymatic kit (Erba Diagnostic Germany Limited, Baroda, India). 
Principle: Alkaline phosphatase (ALP) catalyses the hydrolysis of p-
nitrophenyl phosphate at pH 10.4, liberating p-nitrophenol and phosphate, 
according to the following reaction:  
        p-Nitrophenyl Phosphate + H
2
O   
ALP , Mg2+
 
 
  p-Nitrophenol + Phosphate. 
The rate of p-nitrophenol formation, measured photometrically, is proportional 
to the catalytic concentration of alkaline phosphatase present in the sample.  
Procedure:  
  
Pipette Volumes 
Working reagent* 
1000µl 
Test (Serum) 
20 µl 
*Working reagent: add the amount of Aqua-4 indicated on the label to 
contents of each vial, swirl to dissolve. Mix well and allow standing for 60 
seconds and reading the O.D. against purified water on Photometers at 405nm, 
take the 3 reading with the interval of 60 seconds. 
Calculation: The general formula for converting absorbance change into 
International Units (IU) of activity is: 
 
                     
=   (  A/ min) × T.V × 10

         
 
 
        S.V × Absorptivity × P  
 
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