INTRODUCTION:

In various developed and developing countries about 70% of the population depend on medicinal plants which contribute in the treatment of various maladies and illnesses such as ant diabetics, antiarthritics, hepatoprotectives, cough remedies etc. [1].

It has come to know from various sources that study of herbs is 5000 years old belongs to ancient Sumerians in which a suitable method has described the uses of the medicinal plant. Charaka and Sushruta ancient Indian herbalists had described the various herbs and minerals used in Ayurveda system of medicine. First pictorial evidence of herbal medicine was identified using radiocarbon-dated method between 13000 to 25000 B.C on the wall of the Lascaux caves in France [2]. The pomegranate use extensively in various folk medicines of many cultures. Pomegranate flourished in various parts of world. It is a symbol of fecundity and divine femininity. The word Pomegranate derived from the Latin word Pomum meaning apple and granatus meaning full of seeds. However the botanical name is derived from old French word; Pomegrenate- means Pomegranate apple it belongs to family Lythraceae. Punica granatum is an average height tree mainly grows in India, Iran China-Japan Russia and USA and some parts of eastern countries. It is very much healthy for human being in the treatment of various diseases [3]. Pomegranate has been used by a different culture and civilization from thousands of year ago for treatment of the various ranges of diseases. It is useful in the treatment of skin cancer, osteoarthritis, diabetes, sore throat, coughs, urinary infection, digestive disorder, skin disorders, and arthritis. In clinical research it has been found that if pomegranates are taken as a part of a healthy diet it will be helpful in preventing heart disease, heart attacks and strokes because pomegranates makes the blood thin, increase blood flow to the heart, reduces blood pressure, reduces plaque in the arteries, reduces bad cholesterol and increase good cholesterol [4].

Figure1: Picture of Punica grantaum plant taken at herbal garden of PSIT campus.

Botanical Description:

Pomegranate is a small tree height measuring from five to eight meters, it is important endemic plants of Iran grows in semiarid and arid regions as it has the ability to adapt to adverse ecological condition. It can be divided in to several anatomical compartments including seed, juice, peel, leaf, flower, bark and root each of them contains many pharmacological and toxicological activities. According to holy book of Muslims that is Quran pomegranates grow in the garden of Paradise and Quran has recited pomegranate as an of gods good work [5]. In various researches it has been found that pomegranate has two main species P. protopunica Balf and P. granatum L [6] Pomegranate tree has twisted branches with thorns and red-gray bark, leaves are mostly opposite oblong, deciduous, shiny and stiff. Flower blossom in the month of May to July at the end of the branches. Fruit is round in shape with yellow and orange in color and in berry shape. Fruit is divided internally in to 7-15 cavities embedded with seeds having sweet and sour pulp, transparent and juicy. Not only for health issue even this plant and its parts such as fruits and flower are used for decoration of tables and dishes purpose also. There are three types of anar according to taste sweet anar, sour anar, and combination of both sweet and sour [8].

Figure 2: Leaves of Punica granatum collected from herbal garden of PSIT campus

Table 1: Vernacular name of Punica granatum

Language	Name
Sanskrit	Dadimah
Assamese	Dalim
Bengali	Dalimgachh
English	Pomegranate
Latin	Punica grantatum
Punjabi	Anar, dhalim, dharimb
Hindi	Anar
Urdu:	Anarmitha
Malayalam	Dadiman
Marathi	Dalimba
Oriya	Dalimbo, Dalimo
Tamil	Kalumal madalai, Madulam, Madulangam
Telugu	Didymium, Marimba

Table 2: Botanical classification of Punica granatum

Kingdom	Plantae
Domain	Eukarya
Subkingdom	Magnaliophyta
Division	Magnoliophyta
Class	Magnoliopsida
Order	Myrtales
Family	Punicaceae
Genus	Punica l.
Species	Granatum

MATERIAL AND METHOD:

The fresh leaves of a plant of Punica grantaum L was collected in the month of August – September from the PSIT College of pharmacy campus the plant was authorized by Rashmi Saxena Pal Assistant Professor of Pharmacognosy in PSIT. The leaves were washed with tap water, all dust and unwanted things get washed than the leaves were allowed to dry under the shaded condition or air dried for 7-14 days. After 14 days when leaves are fully dried than fine powder was made using mixer grinder. Extraction was done using ethanol as solvent, solvent was evaporated to dryness and the obtained extract was stored in air tight container at 4°C. For phytochemical screening same ethanol extract was used [3].

Pharmacognostic Evaluation:

Macroscopic characteristic:

Fresh leaves approx. 2.5 to 3.5 cm in length were used for morphological observations. Morphological features of leaves were observed under the magnifying lens [3].

Powder study: The powder of Punica granatum Leaf was light greyish green in color with characteristic odor and astringent taste.

Physicochemical Parameters:

In this section parameters such as Determination of foreign matter, loss on drying, total ash value, and the extractive value was determined in a proper scientific procedure such screening method gives the idea about the nature of the chemical constituents present in a sample. The screening was done according to the method and procedure as given in Khandelwal [9].

Determination of foreign matter (in whole):

Foreign matters are of two types’ organic foreign matter (moulds, insects, animal excreta etc.) and Inorganic foreign matter (stone, soil, dust, sand etc.). 250g of leaves were taken and spread in a thin layer, foreign material was sorted out by visual inspection and by using sieve number 44. The remaining sample was passed through a sieve number 250 to remove dust the remaining obtained sample is free from all impurities [14].

Determination of moisture (loss on drying):

Moisture was determined by loss on drying method It comes under the gravimetric method, in it 1.5 to 2 gm. of powder drug was taken in to thin and flat porcelain dish. Which was allowed to dry in the oven at 100 ̊C to 105 ̊ C, until two consecutive weighing do not differ by more than 0.5mg. Allowed to cool in a desiccator. The loss in weight was usually recorded as moisture. The burning of the powder was proceed slowly and the material must not be allowed to catch fire or to give off smoke and dense fumes. The desiccation method was useful for materials that melt to a sticky mass at elevated temperatures. The loss in weight was recorded as moisture

Determination of ash value of a crude drug:

Ash value was used to determine quality and purity of a crude drug. Ash contains inorganic radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium etc. quantitative determination of ash value helps in their standardization. 3gm powder drug was taken in tarted silica crucible. Incineration was done using Muffer Furnace at 400to 420̊c temperature until it free from carbon and cool. Kept in a desiccator, ash was weighted and percentage of total ash was calculated with reference to the air dried sample of crude drug.

Acid insoluble ash:

Total ash obtained, was boiled for 5 min with 25 ml of dilute hydrochloric acid filtered with ash less filter paper and material was collected on ash less filter paper hot water was poured above it and was washed ignited in crucible cool and was kept in desiccator the obtained residue was weighted and acid insoluble ash was calculated and compared with air dried drug.1.5 gm. was the obtained acid insoluble ash.

Extraction (soxhlet extraction):

Soxhlet apparatus was set up for the extraction of Punica granatum the solvent used in the extraction was ethanol. Gradually 400 ml was ethanol was added to the powder of the punica granatum in the Soxhlet apparatus. Three cycles were allowed to run until the clear liquid was collected in the round bottom flask. It took 72 hours for the completion of three cycles. Thus the extract was collected in the round bottle flask and it concentrated in the Rota Vapor till 25 ml was left in the round bottom flask. Phytochemical analysis of the extract was done to confirm the presence of drug in the extract. The non -soluble portion of the extracted solid remains in the thimble, and is usually discarded [9]

Figure 3: Picture of soxhlet apparatus contains crushed leaves of Punica granatum and ethanol as solvent.

Fluorescence Analysis:

When the sample was exposed to ultraviolet radiation many crude drugs exhibit the phenomenon of Fluorescence. If the substances themselves are not fluorescent, they may often be converted into fluorescent derivatives by applying different reagents. The changes in appearance and color with freshly prepared special reagents under standard conditions was observed and recorded we can often assess qualitatively some crude drugs using fluorescence as it is the most important parameter of pharmacognostical evaluation. Powder gives pale green, green, light brown and brown color. [13]

Rheology:

Rheology is the branch of physics that deals with the flow of matter in this, the study of the coarsely powdered leaf of crude drug was studied in different parameters. Bulk density, angle of repose, compressibility, Hausner's ratio were calculated according to standard procedure.

Bulk density:

The bulk density of a powder was the ratio of the mass of an untapped powder sample and its volume including the contribution of the inter particulates voids volume. Hence, the bulk density depends on both the density of powder particles and the spatial arrangement of particles in the Powder bed. 10g of powdered drug was taken in a graduated measuring cylinder and tapped on a wooden surface. Bulk density is calculated by using the formula [15]

Bulk density = weight taken / Bulk volume

Tapped Density:

The tapped density was obtained by mechanically tapping a graduated measuring cylinder or Vessel containing the powder sample. After observing the initial powder volume or mass, the Measuring cylinder or vessel was mechanically tapped, and volume or mass readings are taken until little further volume or mass change was observed. Devices that rotate the cylinder or vessel during tapping may be preferred to minimize any possible separation of the mass during tapping down. Tapped density was measured by tapped apparatus [15].

Angle of Repose:

The angle of repose or critical angle of repose of a granular material is the steepest angle of descent or dip relative to the horizontal plane to which a material can be piled without slumping. At this angle, the material on the slope face is on the verge of sliding. The angle of repose can range from 0° to 90°.To measure the angle of repose funnel method was used. The powder was allowed to flow through a funnel which was fixed on a stand to form a heap. Height and radius are required to measure the angle of repose. [16]

Compressibility / Carr’s Index:

The Carr's index or Carr's Compressibility Index is an indication of the compressibility of a powder. It is named after the scientist Ralph J. Carr. Cars index greater than 25 is considered to be an indicator of poor flow ability and below 15 is of good flow ability [17].

Hausner’s Ratio:

The Hausner ratio is a number that is correlated to the flow ability of a powder or granular material. It is named after the engineer Henry H. Hausner (1900–1995). Hausner ratio greater than 1.25 is considered to be an indication of poor flow ability [10].

Phytochemical Screening:

Phytochemical Screening is very important as Most of the pharmacological activities possessed by the crude drugs are attributed to the presence of active principles. The observations showed the presence of alkaloids, carbohydrate, reducing sugar, phenol, proteins, coumarins, steroids, tannins, flavonoids, and triterpenoids.

Test for alkaloids:

Wagner's reagent was added to 0.5 ml of plant extracts, A reddish brown precipitate confirms that thetest as positive.

Test for carbohydrate:

0.5gm α- naphtol was addedto 1 ml of extract, from side of test tube concentrated H2SO4 was addedthe presence of violet ring confirms the presence of carbohydrate.

Test for reducing sugar:

1 ml of plant extract was mixed with few drops of Benedict’s reagent and was kept in boiling water bath, observed for the formation of reddish brown precipitate. A positive result shows the presence of reducing sugar.

Test for flavonoids:

0.5 ml of plant extracts was taken, few drops of sodium hydroxide solution was added. A yellow coloration which turns to colorless by addition of few drops of dilute acetic acid indicated the presence of flavonoids.

Test for phenols:

A portion of the extract was treated with aqueous 5% ferric chloride and formation of deep blue or black color indicates the presence of phenols.

Test for proteins:

1 ml of distilled water was added to the extract which was then heated with Biuret reagent and observed for the formation of violet/pink color.

Test for coumarins:

Few drops of 10% NaOH were added to 2 ml of the test solution, appearance of yellow color indicates the presence of coumarins.

Test for steroids:

1 ml of extract was treated with few drops of chloroform, acetic anhydride and conc. H2SO4 and formation of dark pink or red color indicated the presence of steroids.

Test for tannins:

2 ml of extract was treated with 10% alcoholic ferric chloride solution and formation of blue or greenish color solution indicated the presence of tannins.

Test for terpenoids:

1 ml of chloroform was added to 2 ml of each extract followed by a few drops of concentrated sulphuric acid. A reddish brown precipitate produced immediately indicated the presence of terpenoids.

RESULT AND DISCUSSION:

Macroscopic characteristics:

When the macroscopic character was seen it has been observed that leaf was simple in composition, opposite and decussate. The average size of the leaf was 4 to2 cm in length and 2 cm (width). The color of the leaf was observed as green.

Table 3: Organoleptic features of Punica granatum leaves

Serial No.	Features	Result
1	Length	2-4cm
2	Width	2cm
3	Color	Green
4	Shape	Opposite
5	Appearance	Shiny
6	Odor	Characteristic

Physiochemical Properties:

Physiochemical properties of powder of Punica granatum leaves such as loss on drying, total ash, acid insoluble ash and pH was done by using the method as mentioned above. The result obtained are shown in the below given table.

Table 4: Physiochemical properties of powder of Punica granatum leaves

S. No.	Parameter	Result % (W/W)
1	Loss On Drying	17.04%
2	Total Ash	4.66%
3	Acid Insoluble Ash	1%
4	pH	4

Fluorescence Analysis:

Fluorescence analysis of powder of leaves of Punica granatum was done using different chemical such as HCL, Methanol, Ethyl acetate and Benzene which shows different color at different wavelength such as day light, short wavelength and longer wavelength. The result obtained in fluorescence analysis were shown as below in the table 5.

Table 5: Result showing the Fluorescence analysis of powder of leaves of Punica granatum

S. No.	Reagent	Color (Day Light)	Color(Short Wavelength 254nm)	Color(Long Wavelength 365nm)
1	Powder	Pale Green	Dark Green	Pale Green
2	Powder + Water	Green	Dark Brown	Dark Brown
3	Powder + HCL	Greenish Brown	Pale Brown	Brown
4	Powder +Methanol	Green	Dark Green	Dark Brown
5	Powder +Ethyl Acetate	Light Green	Dark Green	Dark Brown
6	Powder +Benzene	Light Brown	Light Green	Dark Brown

Rheological parameters:

Rheological parameters such as Bulk density, Tapped density, Angle of repose, Carr’s index and Hausners ratio was determined by using the above given method and the result obtained were shown in below given table.

Table 6: Rheological parameters of powder of Punica granatum leaves

Serial No.	Rheological Parameters	Result
1	Bulk Density	0.55
2	Tapped Density	0.66
3	Angle Of Repose	5
4	Compressibility /Carrs Index	16.66
5	Hausner’s Ratio	1.22

Phytochemical screening:

Table 7: Phytochemical screening of ethanolic extract of crushed leaves of Punica granatum.

Serial No	Phytochemical Constituent	Aqueous Extract	Ethanolic Extract
1	Alkaloids	+	+
2	Carbohydrate	+	+
3	Reducing Sugar	+	+
4	Flavanoida	+	+
5	Phenols	+	+
6	Proteins	+	+
7	Coumarins	+	+
8	Steroids	+	+
9	Tannins	-	+
10	Terpenoids	+	+

+ Sign indicates the presence of chemical substance whereas – sign indicates the absence of that particular chemical constituent.

CONCLUSION:

Pomegranate is useful in treatment of various diseases, many people believe in herbal treatment as they are very same and effective. The study could be useful in diagnostic for the identification and preparation of the monograph of plant. Various physicochemical parameters were established which can be important in detecting adulteration and mishandling of the crude drug. Medicinal plant Punica granatum is the source of the secondary metabolites such as carbohydrates, glycosides protein, tannins, triterpenoids and flavonoids. The phytochemical analysis of Punica granatum was done. Since the ethanolic extract of P. granatum leaves contains more constituents it can be considered beneficial for further investigation. A typical research and developmental work needs to be carried out for its better therapeutic and commercial utilization. Despite the currently available anti-diabetic drugs, there is need for alternatives which are economical and safe. Many Indian medicinal plants recommended for the treatment of diabetes mellitus lack rigorous scientific justification.

ACKNOWLEDGEMENT:

My sincere thanks to Director Pharmacy Dr. A.K Rai and Department of Pharmacy at PSIT who provided the facilities to carrying out my Research work successfully.

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Received on 14.01.2019 Modified on 14.02.2019

Research J. Pharm. and Tech. 2019; 12(6): 2832-2837.

DOI: 10.5958/0974-360X.2019.00477.3