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                                                                                                           Review of Literature 
that the preventive administration of Opuntia ficus-indica extracts may be helpful in 
alleviating the excitotoxic neuronal damage induced by global ischemia. 
 
12.
 
Antispermatogenic 
A methanolic extract from O. dillenii Haw. defatted with chloroform and petroleum ether 
exerted antispermatogenic effects in animal tests on rats. According to (Gupta et al., 
2002), the flavone derivatives vitexin and myricetin were found to be the active 
principles. When 250 mg extract per kg body weight was applied, the weight of testis, 
epididymis, seminal vesicle, and ventral prostate were reasonably, that of Sertoli cells, 
Leydig cells, and gametes considerably reduced. The motility of the sperms was also 
diminished. 
 
13.
 
Wound healing 
In traditional medicine extracts of polysaccharide-containing plants are widely employed 
for the treatment of skin and epithelium wounds and of mucous membrane irritation. The 
extracts of Opuntia ficus-indica cladodes are used in folk medicine for their antiulcer and 
wound-healing activities. The methanolic extract of Opuntia ficus-indica stems and its 
hexane, ethyl acetate, n-butanol and aqueous fractions (100 mg/site) exhibited wound 
healing activity in rats by measuring the tensile strength of skin strips from the wound 
segments. The extract and less polar fractions showed significant effects (Park & Chun, 
2001). 
Trombetta et al. (2006) described the wound-healing potential of two lyophilized 
polysaccharide extracts obtained from O. ficus-indica (L.) cladodes applied on large full-
thickness wounds in the rat. The wound-healing effect is more marked for 
polysaccharides with a molecular weight ranging 10
4
–10
6
 Da than for those with 
molecular weight>10
6
 Da, author supposed that the fine structure of these 
polysaccharides and their particular hygroscopic, rheologic and viscoelastic properties 
may be essential for the wound-healing promoter action. 
 
 
 
 
85

                                                                                                           Review of Literature 
14.
 
Monoamino-oxidase inhibition 
Besides catecholmethyltransferases, the monoamino-oxidases (MAOs) are usually 
involved in the catabolism of catecholamines, thus regulating the overall amine pool. In 
cladodes and fruits from the Korean O. ficus-indica var. saboten Makino, methyl esters 
derived from organic acids were identified as MAO inhibitors. The aqueous extracts 
showed least inhibitory activity, followed by the n-butanol fraction and the hexane 
extract whereas the ethyl acetate fraction exerted the highest inhibitory action. The active 
agents were identified as 1-methyl malate, 1-monomethyl citrate, 1,3-dimethylcitrate, and 
1,2,3-trimethylcitrate. The purified components showed MAO-A inhibitory action with 
increasing number of methyl substituents, whilst the MAO-B inhibitory action was 
superior for 1-methylmalate compared to the mono- and dimethylcitrates. However, 
1,2,3-trimethylcitrate exerted the strongest inhibition on both MAOs. When citrate was 
compared with its corresponding methyl derivatives, the methoxy moiety proved to be 
the effective moiety (Han et al., 2001). 
 
15.
 
Nutritional important 
Cacti have long been considered an important nutritional source in Latin America (bread 
of the poor) among which Opuntia has gained highest economic importance worldwide. 
It is cultivated in several countries such as Mexico, Argentina, Brazil, Tunisia, Italy, 
Israel and China. Both fruit and stems have  been  regarded  to  be  safe  for  food 
consumption. The constantly increasing demand for nutraceuticals is paralleled by a more 
pronounced request for natural ingredients and health-promoting foods. The multiple 
functional properties of cactus pear fit well this trend. Recent data revealed the high 
content of some chemical constituents, which can give added value to this fruit on a 
nutritional and technological functionality basis. High levels of betalains, taurine, 
calcium, magnesium, and antioxidants are noteworthy (Piga, 2004; Stintzing & Carle, 
2005; Feugang et al., 2006). 
 
The  Opuntia  species cladodes and fruits serve as a source of varied number of 
phytoconstituents mainly sugar, phenolics and pigments. Total betalains are well reported 
with their qualitative and quantitative analytical methods. Though various analytical 
 
86

                                                                                                           Review of Literature 
methods are reported, but still some focus is required towards HPTLC with marker’s 
evidence. Although the reported evidences provide the effectiveness of Opuntia species, 
but active constituents, bioavailability, pharmacokinetics and physiological pathways for 
various biological actions are not well known with sufficient detail or confidence. 
Ethnopharmacological actions may be due to presence of phenolics and pigments. Still 
more attention is required towards the development of simple, feasible and cost effective 
pharmaceutical preparations of Opuntia spp. cladodes and fruit juice as well as the 
ethnopharmacological approach, if combined with mechanism of action, biochemical and 
physiological methods, would provide useful pharmacological leads. 
 
2.4 Research envisage
 
Present study aiming to study phytochemical and pharmacological screening of fruits of 
Opuntia elatior Mill., in Gujarat, commonly known as “Hathlo Thor” belongs to sub-
family Opuntioideae of the family Cactaceae. The literature study reveals that still today 
there is no record of phytochemical composition and pharmacological study of Opuntia 
elatior Mill. fruits in support of traditional and folkloric use. 
 
The Present Project Deals with the Following study: 
1.
 
Collection of fresh plant (Opuntia elatior Mill.) from field and study 
morphology. 
2.
 
To authenticate the plant (Opuntia elatior Mill.) by the Government 
Herbarium Authority. 
3.
 
To study morphology of different parts of the plant.  
4.
 
To evaluate physical parameters of fruit juice. 
5.
 
To prepare different extracts of fruit peel and juice of fruit pulp, and screen to 
detect different types of phytoconstituents using chemical tests and thin layer 
chromatography. 
6.
 
To estimate different types of phytoconstituents using various instrumental 
methods. 
7.
 
To screen fruit juice for antiasthmatic and haematinic activity using different 
experimental models. 
 
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                                                                                                           Review of Literature 
8.
 
To screen different extracts of fruit peel for antimicrobial activity. 
9.
 
Estimation of various haematological parameters like haemoglobin content, 
total red blood cells (RBC), total white blood cells (WBC), differential white 
blood cells counts, 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) and biochemical parameters like 
blood sugar, creatinine, urea, alkaline phosphatase, bilirubin, total Protein, 
total cholesterol, and triglycerides during pharmacological screening.  
10.
 
Histopathological study to conform pharmacological activities. 
 
The Promising Aspect of the Project: 
The Opuntia elatior Mill. is xerophytic wild plant. The plant can grow automatically in 
the desert area and virgin soil without any extra efforts. It does not require any 
maintenance to survive. Raw material of this plant is highly cheaper and easily available 
without any extra burden; in short it is highly economical. Even though easy, wide and 
cheap availability of Opuntia elatior Mill., it is not used in the medicine because 
therapeutic efficacy of this plant is not checked still today. Our aim is to investigate 
therapeutic worth of Opuntia elatior Mill. so that local community and common man can 
explore benefits of this plant. Ultimate our aim is  
1.
 
To generate pharmacological data of Opuntia elatior Mill. in the support of 
traditional and folkloric use. 
2.
 
This study can inspire poor people to use easily available cheaper plant as a 
medicine.  
3.
 
To generate morphological, physicochemical and phytochemical data to know 
the identity, purity and quality of the plant. 
 
88

 
 
 
3. Materials and Methods 
 

3. Materials and Methods 
Sr No. 
Title 
Page 
No. 
3.1 Apparatus 
89 
3.2 Chemicals 
90 
3.3 Pharmacognostical 
studies 
90 
3.3.1 
Collection and authentication of plant 
90 
3.3.2 Macroscopic 
examinations 
90 
3.3.3 Proximate 
analysis 
91 
3.3.3.1 
Determination of average weight, % of peel, pulp 
and seeds of fruit 
91 
3.3.3.2 
Preparation of fruit juice of Opuntia elatior Mill. 
(OFJ) 
91 
3.3.3.3 
Determination of pH of OFJ 
91 
3.3.3.4 
Determination of Moisture content and Total 
solids 
91 
3.3.3.5 
Determination of Ash value  
92 
3.3.3.6 
Determination of Density of OFJ 
92 
3.3.3.7 
Determination of Viscosity of OFJ 
92 
3.4 Phytochemical 
studies 
93 
3.4.1 
Preparation of fruit peel extracts 
93 
3.4.2 Qualitative 
evaluation 
of 
peel extracts and OFJ 
94 
3.4.3 
TLC profile of OFJ 
94 
3.4.4 
Qualitative analysis of betalain 
94 
3.4.4.1 Spectrophotometric 
analysis 
94 
3.4.4.2 
High performance liquid chromatographic 
(HPLC) analysis 
95 
3.4.4.3 Liquid 
chromatography – mass spectroscopic 
(LC-MS) analysis 
95 
3.4.5 
Quantitative estimation of OFJ 
96 
3.4.5.1 Total 
sugar 
content 
96 

3.4.5.2 
Total phenolic content 
96 
3.4.5.3 Titratable 
acidity 
97 
3.4.5.4 
Total betacyanin content 
97 
3.4.5.5 Elemental 
analysis 
97 
3.5 Pharmacological 
studies 
99 
3.5.1 Plant 
material 
99 
3.5.2 Animals 
99 
3.5.3 
Acute toxicity study 
99 
3.5.4 Haematinic 
action 
100 
3.5.4.1 HgCl
2
-induced anaemia 
100 
3.5.4.1.1 Experimental 
design 
100 
3.5.4.1.2 
Measurement of Body weight 
101 
3.5.4.1.3 
Measurement of Haematological Parameters 
101 
3.5.4.1.4 
Measurement of Biochemical Parameters 
102 
3.5.4.1.4.1 
Estimation of Blood Sugar 
102 
3.5.4.1.4.2 
Kidney functions study 
103 
3.5.4.1.4.2.1 
Estimation of Creatinine 
103 
3.5.4.1.4.2.2 
Estimation of Urea 
103 
3.5.4.1.4.3 
Liver functions study 
104 
3.5.4.1.4.3.1 
Estimation of Alkaline phosphatase (ALP) 
104 
3.5.4.1.4.3.2 
Estimation of Bilirubin 
105 
3.5.4.1.4.3.3 
Estimation of Total Protein 
106 
3.5.4.1.4.4 
Estimation of Total Cholesterol 
106 
3.5.4.1.4.5 
Estimation of Triglyceride  
107 
3.5.4.1.5 
Histopathology of Liver, Kidney and Spleen 
108 
3.5.4.2 Phenylhydrazine-induced 
anaemia 
108 
3.5.4.2.1 Experimental 
design 
108 
3.5.4.2.2 
Measurement of Body weight 
109 
3.5.4.2.3 
Measurement of Haematological Parameters 
109 
3.5.4.2.4 
Study of the Reticulocytes 
109 
3.5.4.2.5 
Measurement of Biochemical Parameters 
109 
3.5.4.2.6 
Estimation of Ferritin 
110 

3.5.4.2.7 
Histopathology of Liver, Kidney and Spleen 
111 
3.5.5 Antinociceptive 
tests 
112 
3.5.5.1 Writhing 
test 
112 
3.5.5.2 
Tail immersion test 
112 
3.5.6 Anti-asthmatic 
action 
113 
3.5.6.1 
Bronchospasm induced by Acetylcholine and 
Histamine in guinea pigs 
113 
3.5.6.2 
Anticholinergic action on isolated rat ileum 
113 
3.5.6.3 
Antihistaminic action of isolated Guinea pig 
ileum 
114 
3.5.6.4 
Egg albumin induced mast cell degranulation test 
114 
3.5.6.5 
Compound 48/80 induced mast cell 
degranulation 
115 
3.5.6.6 
Carrageenan-induced rat paw edema 
116 
3.5.6.7 
Neutrophil adhesion test 
117 
3.5.7 Statistical 
analysis 
117 
3.6 Estimation 
of 
antimicrobial action of fruit peel 
extracts 
118 
3.6.1 Test 
microorganisms 
118 
3.6.2 
Preparation of test organism suspension 
118 
3.6.3 Antimicrobial 
assay 
119 
 

                                                                                      Materials and Methods 
3. Materials and Methods 
3.1 Apparatus 

 
A double beam atomic absorption spectrophotometer (AAS) (AA-
6300, Schimadzu). 

 
ACCULAB digital balance, (Model No. ALC-310.3, Sartorius 
Mechatronics India Pvt. Ltd., Bangalore, India). 

 
Automated fluorescence flow cytometry 5-part different analysers 
(Sysmex XS800i, Japan). 

 
Blender (Boss appliances, Daman, India). 

 
Brookfield viscometer (Model DV-II+ Pro viscometer). 

 
Compufuge cooling centrifuge (Remi Instrument, Mumbai, India). 

 
Density bottle (Borosil Glass Works Ltd., Mumbai, India). 

 
Digital pH meter (model-EQ-610, Equip-Tronics, Ahmedabad, India). 

 
Double beam THERMO UV-visible spectrophotometer (Thermo 
Spectronic, Cambridge, UK) equipped with VisionPro software V 
4.10. 

 
Glass filter G
4
 (Borosil Glass Works Ltd., Mumbai, India). 

 
High performance liquid chromatography (Shimadzu). 

 
Liquid chromatography – Mass spectroscopy (TSQ Quantum Ultra, 
Thermo Scientific, USA). 

 
Muffle furnace (Janki Impex, Ahmedabad, India). 

 
Sahli’s haemoglobinometer (Janki Impex, Ahmedabad, India). 

 
Silica gel 60 F 254 precoated plates (Alugram
®
 SIL G/UV
254

Macherey – Nagel, Germany). 
 
89

                                                                                      Materials and Methods 
3.2 Chemicals 

 
Acetylcholine, carrageenan, compound 48/80, cresyl blue, gallic acid, 
histamine, mercuric chloride, phenylhydrazine hydrochloride were 
procured from Sigma Aldrich, Mumbai, India. 

 
Acetic acid, acetonitrile, anhydrous citric acid, anthrone reagent, 
benzene, chloroform, concentrated sulfuric acid, deionized water, 
distilled water, Folin-Ciocalteu’s reagent, glucose, hydrochloric acid, 
methanol, nylon fibers, petroleum ether (60-80
0
C), sodium carbonate, 
sodium hydroxide, toluidine blue were procured from S.D Fine 
Chemicals Mumbai, India. 

 
Muller Hilton agar, Potato Dextrose agar were purchased from 
Himedia Lab., India. 

 
Diagnostic kits used for estimation of blood sugar (Bayer diagnostics, 
Ahmedabad, India), serum urea, creatinine, total cholesterol, 
triglyceride (Nicholas India Pvt. Ltd., Ahmedabad, India), alkaline 
phosphate, bilirubin (Erba diagnostic Germany Ltd., Baroda, India) 
and total protein (Span diagnostics India Pvt. Ltd., Ahmedabad, India). 
 
3.3 Pharmacognostical studies 
3.3.1 Collection and authentication of plant 
The fruits of Opuntia elatior Mill. were collected from road side weed near 
Atkot, Ta: Jasdan, Dist: Rajkot, Gujarat, India at Latitude (22
0
 1’ 48” N), 
Longitude (71
0
 12’ 0” E) and Elevation 193 M (633 ft) and authenticated by 
Dr. H. B. Singh, Scientist and Head, Raw Materials Herbarium and Museum, 
National Institute of Science and Communication and Information Resources, 
New Delhi (NISCAIR) and preserved the herbarium (specimen voucher No.: 
rbpmpc/museum/herbarium/07-08/01)  in the museum of  Dept. of 
Pharmacognosy, Smt. R. B. Patel Mahila Pharmacy College, Atkot. 
3.3.2 Macroscopic examinations 
The whole plant of Opuntia elatior Mill. was subjected to macroscopic 
examinations using reported methods in standard text (Datta, 2003) and the 
results were compared with the reported monographs (Kirtikar and Basu, 
1999; The Wealth of India, 2001). 
 
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                                                                                      Materials and Methods 
3.3.3 Proximate analysis 
3.3.3.1 Determination of average weight, % of peel, pulp and seeds of fruit  
Mature fruits (50 units) of Opuntia elatior Mill. were collected and taken 
immediately to the laboratory where they were weighed before and after 
manually peeled out, subjected to homogenization, and separated seeds were 
again weigh using ACCULAB digital balance, Model No. ALC-310.3 
(Sartorius Mechatronics India Pvt. Ltd., Bangalore). 
 
3.3.3.2 Preparation of fruit juice of Opuntia elatior Mill. (OFJ)  
Mature fruits of Opuntia elatior Mill. were collected and immediately taken to 
the laboratory. Spines and glochides were removed from fruits by just heating 
on the burner and then washed with water. The peel of the fruits was removed 
manually and pulp subjected to homogenization for 5 minute using boss 
portable blender (Boss appliances, Daman). After homogenization, fruits juice 
was filtered though muslin cloth and filtered juice was used for various 
estimation and biological studies. 
 
3.3.3.3 Determination of pH of OFJ  
The pH of OFJ was determined five times using pH meter (Digital pH meter 
model-EQ-610, Equip-Tronics, India) (Anonymous, 1996). 
 
3.3.3.4 Determination of Moisture content and Total solids  
Estimation of moisture content and total solids was carried out five times as 
per Anonymous (1989). Fruit pulp (10 g) placed in a tarred evaporating dish 
and dried at 105 ºC in an oven at constant weight. The moisture content and 
total solids were determined using following equation. 
% Moisture content = [(initial weight – dried weight)/initial weight] X 100 
% Total Solids = (Dried weight / Initial weight) X 100 
 
 
 
 
 
 
 
91

                                                                                      Materials and Methods 
3.3.3.5 Determination of Ash value  
Total ash, acid-insoluble ash and water-soluble ash values were determined 
five times as per Anonymous (1989) and WHO (2002). 
Total ash:  3g of accurately weighed fruit pulp was taken in a tarred silica 
crucible and incinerated at a temperature not exceeding 450 ºC until free from 
carbon and constant weight, cooled and weighed.   
Acid-insoluble ash: Total ash obtained was boiled for five minutes with 25 ml 
of dilute Hydrochloric acid. The insoluble matter was collected on an ash less 
filter paper, washed with hot water and ignited, cooled and weighed.  
Water-soluble ash: Total ash obtained was boiled for five minutes with 25 ml 
of distilled water, cooled and collect the insoluble matter on an ash-less filter 
paper, washed with hot water and ignited for 15 minutes at temperature not 
exceeding 450 ºC.  
 
3.3.3.6 Determination of Density of OFJ  
Density of OFJ was determined five times using density bottle at room 
temperature against water as reference compound (Gaud and Gupta, 2006). 
 
3.3.3.7 Determination of Viscosity of OFJ 
Viscosity of OFJ was determined five times using spindle S61 of Brookfield 
viscometer (Model DV-II+ Pro viscometer) at 100 rpm (Gaud & Gupta, 2006). 
 
92

                                                                                      Materials and Methods 
3.4 Phytochemical studies 
3.4.1 Preparation of fruit peel extracts 
Manually removed peel of fruits was subjected to air drying at room 
temperature. Air dried peel was pulverized and passed through 10 # sieve. A 
finely peel powder was extracted successively with petroleum ether (60-80
0
C), 
benzene, chloroform, methanol and water in Soxhlet extractor for 24 hours 
(Scheme – I).  
 
Powdered peel (100 gm) 
           Extracted with petroleum ether (60-80
0
C) 
 
 
 
                      (750 ml) in Soxhlet apparatus for 24 hours 
 
Petroluem 
ether 
extract     Residue 
     (Air-dried, 
reweighed, 
repacked) 
 
 
 
 
 
 
        Benzene (750 ml) 
 
Benzene extract 
 
 
 
 
 
     Residue 
     (Air-dried, 
reweighed, 
repacked) 
 
 
 
 
 
 
       Chloroform (750 ml) 
 
Chloroform 
extract 
      
 
 
 
 
Residue 
     (Air-dried, 
reweighed, 
repacked) 
 
 
 
 
 
 
 
Methanol (750 ml) 
 
Methanol extract 
 
 
 
 
 
     Residue 
     (Air-dried, 
reweighed, 
repacked)
 
 
 
 
 
 
 
 
     Water (750 ml) 
 
Water 
extract 
       
 
 
 
Residue 
 
 
 
 
 
 
 
          (Discarded) 
 

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