EXTRACTION

EXTRACTION, INVITRO SCREENING AND PREPARATION OF TABLETS FROM DRAGON FRUIT PARTS
INTRODUCTION
The dragon fruit is an epiphyte plant which belongs to the family Cactaceae family. It is principally originated from the tropical and subtropical regions, but their growth is rich in the European countries due to its fruit exotic value. The name of the dragon fruit differs from region to region. For example, the dragon fruit in Israel is called Pitahaya fruit and buah naga in Indonesia. The botanical name of the dragon fruit is called as Hylocereus undatus. Based on nature of stem habit and pulp color the edible dragon fruit is classified into different varieties. The different varieties of dragon fruit are mentioned below:
Hylocereus undatus
Hylocereus triangularis
Hylocereus costaricensis
Hylocereus polyrhizus
Hylocereus megalanthus
TAXONOMY: The genus Hylocereus is a small genus that contains about 18 tropical American species. The name Hylocereus genus is vine (climbing with aerial roots or epiphytic in nature) cacti with three angled stems and mostly with very fragrant nocturnal white flowers. Dragon fruit is a common name for fruits of several cacti species. As these are new crops, their taxonomical identity is not yet known completely it is in confusion.
Fruit morphology: The fruit is a medium to large, oblong-shaped epigenous berry. The berry is distinguished with red skin with large scales. The fruit pulp may be white, red or yellow and juicy depending on the varieties/species.

PROPERTIES OF SPECIES, USES, AND PRODUCTS
Dragon fruit is a nutritious fruit with a variety of uses, although the composition of this species has not been extensively studied, particularly with reference to the components of the fruit. The most valuable and commonly used edible part of Dragon fruit is the fruit pulp which constitutes 70-80% of the ripe fruit. The composition of fruit pulp is given in Table 4.4,
The most valuable and commonly used edible part of Dragon fruit is the fruit pulp which is eaten raw as a fresh fruit. The pulp constitutes 70-80% of the ripe fruit. The flavor of fruit pulp is sometimes Similar to that of the Kiwi fruit. Dragon fruit pulp could be chilled and cut into half to show the attractive flesh, either sliced or scooped with a spoon. It is widely used in the preparation of cakes and salads.

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The nomenclature of Dragon fruit is as follows:
Kingdom Planate (Plants)
Subkingdom Tracheobionta (vascular plants)
Super division Spermatophyta (seed plants)
Division Magnoliophyta (flowering plants)
Class Magnoliopsida (dicotyledons)
Order Caryophyllales
Family Cactaceae (cactus family)
Subfamily Cactoideae
Tribe Hylocereae
Genus Hylocereus.

Species Hylocereus undatus
CHEMICAL CONSTITUENT:
High moisture content
Low protein contents.

Lipid contents.

The total food fiber.
Ashes.

Highest glucide contents.

Contents of vitamin C
Dietary fiber-rich powder from piyata peel (DFRPP)
Oligosaccharides
Dietary fiber,
?-carotene,
Magnesium, calcium, potassium
Mucilage
Total Phenol
Polysaccharides
?4-?-d-GlcpA-1?, ?6-?-d-Galp-1, and ?4-?-l-Rhap-1? constituted the backbone and ?-l-Araf-1 ? 5-?-l-Araf-1? formed the branch chain. The precise structure was putatively identified as below. The molecular weight was 2.2 × 103 kDa
Betacyanin
Pectic substances
The cell wall polysaccharides of the peel, as well as those of the pulp, contain high amounts (38–47 %) of loosely bound (water-soluble) pectic substances.

In the water-soluble fraction (WSF) of the peel samples, uronic acids are the predominant monomers. The rhamnogalacturonan-I type neutral sugars, and especially arabinose and galactose, contribute equally, as compared to uronic acid, to the WSF of the pulp samples.

The wide range of epitopes, including long blocks of unesterified galacturonic acids (GalA) residues as well as (short) blocks of esterified GalA residues
Anthocyanins
Minerals
Plants albumin
Water-soluble dietary fiber
Unsaturated fatty acids
Vitamins
Flavanoids and flavonols
Linoleic acid
Beta-carotene, 3.4 ± 1.4 ?g
Lycopene and 0.26 ± 0.06 mg
Vitamin E (Charoensiri et al., 2009)
Vitamin C
Natural antioxidants
Phenolics, tocopherols, and sterols
50 % of essential fatty acids
Linoleic
Other hydrogen-donating groups (-NH, -SH) or hydroxyl group (OH) in polyphenolic molecules.

Betanins which is an important pigment in pitaya also show imino groups and Hydroxyl groups.

USES OF DRAGON FRUIT:
Antiradical activity
Antibacterial activity
Antioxidant activity
Thickening agent and natural colorant in cosmetic hydrating creams
Encapsulating agent
Hypoglycaemic, diuretic, against heart disease (Argueta, 1994; Ankli et al., 1999), wound disinfectant, tumor dissolution with stem sap (Mendieta & Del Amo, 1981), and dysentery cure
They have pre-biotic characteristics, resistance to acid conditions in the stomach, and partial resistance to human ?-amylase, and they also promote lactobacillus and bifidobacteria (Wichienchot et al., 2010).

High potential as a source of antioxidants and essential fatty acids, with an exceptional level of linoleic acid: 660 g Kg- H. undatus and 480 g Kg
Antimicrobial activity against gram-positive and gram-negative bacteria, but H. polyrhizus extracts have the greater effect
Antihepatotoxic effect
Decreases, in rats, Dyslipidemia (increased and modifiable risk factor for cardiovascular disease, particularly coronary disease due to lipid alteration in the blood) (Mohd et al., 2009).

Abortion prevention
Fertility agent
Inhibits PEP case activity
Antibacterial activity
High antiradical activity
Prevention of cardiovascular and cerebrovascular diseases, modulation of immune function and regulation of hormone levels enzyme product development, the natural edible pigment, potent wine production, seed oil extraction, gel juice, and makeup product development.
Low-cost bio sorbent for the removal of toxic dye, methylene blue.

enhanced the growth of probiotic bacteria inhibited the growth of pathogen and as such can be considered as a functional food ingredient for the functional food industry
The effect in T2DM was not significant but there was a trend towards greater blood glucose reduction with a higher dose
Induce Growth Inhibition and Proapoptotic Effects on Human Cell Lines of Breast Cancer via Downregulation of Estrogens Receptor Gene Expression.

Trans-epidermal water loss regulating an activity
Collagen synthesis stimulating activity
Dragon fruit extract is well recommended to formulate cosmetic products with moisturizing, soothing and re-epithelizing activity.
Polysaccharides
AIM AND OBJECTIVES:
The aim of the study is to prepare and punch the tablets by using the dragon fruit part extracts as a binder.

To perform the Invitro screening for antioxidant and antibacterial activities.

To compare the different concentrations of binder with the synthetic binder and marketed tablets.

To perform the evaluation tests for the tablets prepared by using dragon fruit parts.

LITERATURE REVIEW:
Wee Sim Choo* et.al performed the studies on dragon fruit parts to compare the antioxidant level of two different species by using DPPH assay and ferroin ion chelating activity. These studies showed the pulp and peels of both Hylocereus undatus and Hylocereus polyrhizus has the antioxidant activity. But the analysis showed that the pulp of hylocereus undatus is much better than the pulp of Hylocereus polyrhizus.

*Sengkhamparn, N., et.al studied the Effects of blanching and drying on fiber rich powder from pitaya (Hylocereus undatus) peel. In this study, we found that pitaya peel can be used as a raw material for producing fiber rich powder which was a good source of bioactive compounds. This information provided that the pitaya peels is a valuable material for a manufacture of fiber-rich powder with high antiradical activity.
*Osman,A. et.al studied the Antibacterial property of Hylocereus polyrhizus and Hylocereus undatus peel extracts by using disc diffusion method and micro dilution method . The study showed that all the bacteria tested were sensitive to different extracts from red and white flesh pitaya fruit peels. It can be confirmed that, chloroform extracts of both Hylocereus species peel showed greatest antibacterial activity with H.polyrhizus peel being greater than H.undatus peel.

Naw Juna Paw*et.al studied Effect of Dragon Fruit on glycemic Control in Type 2 Diabetes. Dragon fruit reduces blood glucose level by attenuating fibroblast growth factor-21 resistance and regenerating pancreatic cells. Based on the available evidence, it can be concluded that red dragon fruit may possibly be effective in glycemic control of both type 2 diabetes and be beneficial as an add-on therapy. However, due to the limited available data and poor quality of clinical studies, adequate-power, well-controlled clinical trials are required to further evaluate the clinical application of dragon fruit in these patients.
Phanuphong Chaiwut*et.al performed the studies on Extraction and stability of cosmetic bioactive compounds from dragon fruit peel. This study was designed to extract and evaluate stability of cosmetic bioactive compounds from dragon fruit (Hylocereus undatus) peels. Extractable phenolics content (EPC) assayed by Folin-Ciocalteu method and antioxidant capacity assayed by DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays were employed for comparative evaluation. Result of this study showed that the dragon fruit peel could be used as a source for cosmetic bioactive extraction due to it providing weather antioxidant and colorant capacity.

Thanh L. Pham et.al studied the Antibacterial Properties of Hylocereus undatus against opportunistic pathogens present within the Skin, Oral Cavity and gastrointestinal Tract. The aim of this research project was to explore the antibacterial properties of a particular species of dragon fruit, Hylocereus undatus, using various opportunistic pathogens found within the skin, oral cavity and gastrointestinal tract. The project consisted of the determination of the antibacterial activity, specifically the MIC AND BIC of the dragon fruit pulp extract using standard biochemical tests, Kirby-Bauer antibiotic sensitivity testing, and optical density growth analysis methods. The results of these investigations determined that 100% v/v dragon fruit pulp extract was able to inhibit bacterial growth up to 92%.These results indicate that dragon fruit possessed antibacterial properties against opportunistic pathogens found within the oral cavity and gastrointestinal tract.
PLAN OF WORK:
Literature review.

Collection of fruits and separating their parts.

Drying of parts.
Extraction process.

Phytochemical screening.

Invitro screening.

Comparing the level of activity.

Preparing tablets from dragon fruit parts.

Evaluation of tablets.

Conclusion.

COLLECTION OF FRUITS AND THEIR PARTS:

The dragon fruits are purchased from the local markets and then washed thoroughly with the distilled water.
The fruit parts are then separated manually and subjected for drying under the sunlight and air.

The dried parts (Peel, seeds) are then powdered into fine particles and kept ready for further extraction process.

The pulp is subjected for maceration process in order to obtain the drug extract.

EXTRACTION PROCESS:
The above dried materials especially seed and peel powders are subjected for extraction process.

The extraction process is carried out by using soxhlet apparatus.

The powdered drug is packed and thimble is prepared which is placed in the column.

The flask is filled with the solvent ethanol in order to collect the extract and apparatus is set to run with the help of mortar and electricity for the extraction.

The extraction process is carried out for 48hrs to each part of the dragon fruit separately and the extract is collected by the bottom flask which is present with the ethanol.

The solvent is taken in order to separate and collect the extract of three different parts of dragon fruit. The solvent is subjected for heating to evaporate the solvent so that the extract residue is obtained finally. Thus the extracts from different parts of the dragon fruit are prepared and kept for the phytochemical screening test.

PHYTOCHEMICAL SCREENING:
The three extracts (Peel, pulp and seeds) are tested for the presence of phytochemicals which are responsible for the concerned activity.

The results are shown in the table mentioned below.

TABLE 1:

TABLE 2:

INVITRO SCREENING:
Prepare the stock solution of the given samples as 300mg/ml .Prepare the dilutions of the samples as 1000µg/ml, 2000µg/ml and 4000µg/ml.

Melt the nutrient agar and maintain it at 50°-55°c.

Add1ml suspension of test organism to the medium thoroughly while maintaining temperature at 50°c.

Pour the above mixture into Petri dishes to form layer of about 3mm thickness. Allow the medium to solidify.

Cut the reservoirs with sharp tools such as cork borer. Remove the cylindrical plugs with scalpel or sharp forceps.

Mark the cups as per dilutions and add in each cup the respective dilutions of the sample.

Now add the given samples (seed, peel & pulp extracts) which are diluted from the stock solutions.

Keep the plate carefully in the refrigerator for the diffusion of sample for 20 mins
Incubate all the tubes for 20 -24 hrs at 30°-35.

Observation: Record the size of the inhibition zone against each standard dilution and the unknown dilution. The size is measured in mm with the help of scale.

AGAR PLATE CUP METHOD:
Preparing the dilutions of the samples(seed, peel &pulp extracts) from the stock solution.
Melting the nutrient medium.

Add1ml suspension of test organism to the medium.

Pour the medium into the petri plates. Allow it to solidify
Cut the reservoirs with sharp tools such as cork borer.

Mark the cups as per dilutions and add in each cup the respective dilutions of the sample.
Now add the samples (seed, peel &pulp extracts)
Keep the petri plates in a refrigerator for the diffusion of samples for 20 mins.

Incubate all the plates for 20-24 hrs.
Record the zone of inhibition and compare with standard. The zone of inhibition is measured using scale.
RESULTS:

GRAPH:
MINIMUM INHIBITORY CONCENTRATION:
Micro dilution method was performed to know the MIC for the given samples.

The sample extracts are diluted to eight different concentrations from the stock solution.

To continue the further process we need to subculture the medium.

The nutrient broth is directly inoculated with the two different bacterial strains in the presence of laminar airflow and incubated for 24 hrs 30°-35°c.

After incubation the test tubes containing the subcultures are filled with measured quantity of sample extracts(seed, peel& pulp).

Incubated again for 24 hrs 30°-35°c.

Observe the results carefully.

The clear samples indicate the presence of antibacterial activity where as turbid formation indicates the presence of micro organisms.

ANTIOXIDANT ACTIVITY:
Antioxidants are compounds that inhibit oxidation. Oxidation is a chemical reaction that can produce free radicals, thereby leading to chain reactions that may damage the cells of organisms. Antioxidants such as thiols or ascorbic acid (vitamin C) terminate these chain reactions.

DPPH METHOD:
DPPH(2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in ethanol .This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to colourless ethanol solution. The use of the DPPH assay provides an easy and rapid way to evaluate antioxidants by spectrophotometry , so it can be useful to assess various products at a time.

The percentage of antioxidant activity (AA%) of each substance was assessed by DPPH free radical assay.
The samples were reacted with the stable DPPH radical in an ethanol solution.
The reaction mixture consisted of adding 0.5 ml of sample, 3 mL of absolute ethanol and 0.3 ml of DPPH radical solution 0.1 mM in ethanol.
When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in colour (from deep violet to light yellow) were read Absorbance (Abs) at 517 nm after 100 min of reaction using a UVVIS spectrophotometer. 01’1
GRAPH:

% INHIBITION:

GRAPH:

TABLETS FROM FRUIT PARTS:
To punch the tablets from dragon fruit parts the following are required:
API(Naproxen)
Excipients
Starch /dragon fruit extracts (5%;10%)
Lactose (30%)
Magnesium stearate (5%)
Talc (5%)
Polyvinyl pyrrolidine (5%)
The different parts of dragon fruit extracts are used as natural binder in the preparation of tablets and their drug release rate is compared with the synthetic binder available in market.

The tablets are punched using two different concentrations of natural binders (5% ; 10%).

The process also includes the wet granulation method in order to form small granules used for punching the tablets.

The active ingredient concentration is taken as 250mg (API).

The solvent ethanol is used for wet granulation process.

The punching is performed by using ten’s rotary punching station machine with a die cavity containing 9mm.

PROCESS:
Collect all the ingredients required for the process (API/Excipients).

Weigh all the ingredients accurately.

Wet granulation: Mix all the ingredients thoroughly by using suitable solvent so that a uniform paste is obtained.

Now the paste is subjected for sieving process in order to form granules.

Drying of granules.
Weigh the granules required for punching the individual tablet.

Granules are punched by using Ten’s rotary punching station.

The die cavity with 9mm is used for punching the tablets.
Formation of tablets.

EVALUATION:
The solubility parameters are observed for naproxen drug containing synthetic binder and natural binder.

DISSOLUTION :
Buffer: 0.1 M of a phosphate buffer with a pH of 7.4, 2.62 g/L of monobasic sodium phosphate and 11.50 g/L of anhydrous dibasic sodium phosphate
Medium: 900 mL
Apparatus 2: 50 rpm
Time: 45 min
Dissolution was performed with the marketed naproxen tablets and punched tablets containing the natural binder.

HARDNESS TEST:
Tablet hardness is usually expressed as the load required crushing a tablet placed on its edge.
Hardness is thus sometimes termed the tablet crushing strength. The suitability of a tablet in regard to mechanical stability during packaging and shipment can usually be predicted on the basis of hardness.
Tablet hardness, in turn, influences tablet density and porosity. It may affect tablet friability and disintegration time.
It usually affects drug dissolution and release and it may affect bioavailability.

Name and specification of materials required in hardness test :
MATERIALS SPECIFICATION
Hardness tester Monsanto type
Method:
1. The sliding scale of hardness tester has been set off to zero
2. The tablets have been placed vertically between the two jaws.
3. Force has been applied with the screw thread and spring until the tablets has been fractured.
4. A force of about 4-5 kg is considered to be the minimum for hardness according to The British Pharmacopoeia (Lachman, et al.,2008)
Friability test:
Friability is the property of the tablets to remain intact, when tablets are subjected to a rotatory motion, e.g. during the tablet coating processing of, tablet packaging or transport, as this may cause small particles to abrade from the surface of the tablet. In order to avoid those problems the friability test is performed. The Roche friabilator is the most common type of friabilator which is used to measure friability and is rotated at 25rpm for 4mins. After processing reweigh the tablets. Weight loss indicate as the percent friability and the loss of weight should not more than 1%

RESULTS AND DISCUSSION:
DISSOLUTION: The dissolution test was performed with the marketed tablet naproxen according to the specifications in USP and BP. The average dissolved percentage was calculated 90% that comply with limits.

TABLE 1:
TIME %CDR
5 13.4
10 14.95
15 15.99
20 16.89
30 17.66
40 18.32
45 19.98
60 20.54
90 22.54
120 23.96
150 24.98
180 25.99

GRAPH 1:

DISSOLUTON PROFILE OF DRAGON FRUIT TABLET:
The tablets were punched by using the peel extract of dragon fruit .In this process the peel extract is used as natural binder in order to punch the tablets .The two different concentrations( 5% and 10% ) of natural binders are used. The synthetic binder starch is replaced with the different extracts of dragon fruit .The different types of extracts are peel 5%, peel 10%, pulp 5% ,pulp 10%,seed 5%,seed 10%.The different dissolution profiles were compared and their average dissolved percentages are calculated.

TIME %CDR
15 19.89
30 20.12
40 21.32
45 22.53
60
22.74
120 22.91
150 23.13
180 23.64
240 23.86
300 24.10
360 24.67
420 24.92
480 25.2
TABLE 2:
The average dissolved percentage of tablets which are made from the peel 5% extract of dragon fruit are calculated as 90.7%
GRAPH 2:

TABLE 3: Dissolution profile of peel extract 10%
TIME %CDR
15 19.96
30 20.08
40 20.56
45 20.98
60
21.04
120 22.31
150 22.74
180 23.03
240 23.62
300 23.84
360 24.23
420 24.98
The percentage of cumulative drug release was found to be 89.9%
GRAPH 3:

TABLE 4: Dissolution profile of pulp extract 5%
TIME %CDR
15 18.89
30 19.28
40 19.4
45 19.56
60
19.63
120 19.68
150 20.13
180 21.68
240 23.7
300 23.84
360 25.8
420 25.93
The percentage of cumulative drug release was found to be 93.3%
GRAPH 4:

TABLE 5: Dissolution profile of pulp extract 10%
TIME %CDR
15 19.89
30 20.01
40 20.36
45 21.48
60
22.34
120 22.52
150 23.02
180 23.62
240 23.92
300 24.67
360 25.82
The percent cumulative drug release was found to be 92.9%
GRAPH 5:

TABLE 6: Dissolution profile of seed extract 5%
TIME %CDR
15 19.2
30 19.65
40 19.89
45 20.97
60
21.02
120 22.08
150 23.91
180 24.07
240 24.83
300 25.31
360 25.69
The percent cumulative drug release was found to be 92.4%
GRAPH 6:

TABLE 7: Dissolution profile of seed extract 10%
TIME %CDR
15 19.5
30 19.68
40 19.98
45 20.86
60
21.83
120 22.37
150 22.80
180 23.35
240 23.97
300 24.08
360 24.83
420 25.39
The percent cumulative drug release was found to be 91.4%
GRAPH 7:

PARAMETERS Marketed tablet Peel 5% tablets Peel 10% tablets Pulp 5% tablets Pulp 10% tablets Seed 5% tablets Seed 10% tablets
THICKNESS
(mm) ( n =10) 7.5mm 6.75m 7.2m 5.5mm 6.5mm 7.2mm 6.3mm
HARDNESS
(Kg) ( n =10) 14kg 10kg 12.8kg 15kg 14.2kg 16kg 16.4kg
DISSOLUTION
( %) ( n =5) 93.2% 90.7% 89.95% 93.3% 92.5% 92.4% 91.4%
FRIABILITY
( %) ( n =10) 0.123% 0.257% 0.406% 0.510% 0.623% 0.668% 0.732%
WEIGHT (mg)
( n =10) 701.2mg 750.5mg 658.3mg 650.2mg 710.8mg 680.9mg 580.3mg
The different parameters were evaluated and the results were found to be satisfactory. The The two methods were performed to estimate the antibacterial activity.

The agar plate cup method showed the good antibacterial activity and confirmed that the dragon fruit extracts has antibacterial activity.

The antioxidant activity is more for the seed extract when compared to peel and pulp extract.

presences of phenolics are very important to show the activity of antioxidant property.

The phytochemical screening is performed in detail to confirm the presence of the phenolics in the dragon fruit parts.

Finally the different parts of dragon fruit namely pulp, seeds and peel showed the presence of phenolics and other constituents paving the way to carry out the further project work successfully.

The Invitro screening for the both activities are performed and tablets are punched by using fruit parts and they should be subjected for different evaluation in order to compare the level of activity.

The other tablets should be punched by using various concentrations of extracts and evaluated.
Finally all the tablets with varying concentrations are being compared with marketed tablet naproxen and found that the dragon fruit extract which are used as natural binders are showing good effect as binder such that they can be used for the preparation of sustained release formulations in the upcoming future .so, we can formulate the innovative drugs from this dragon fruit extracts with the antioxidant and antibacterial activities
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