PHARMACOGNOSTIC AND PHYTOCHEMICAL EVALUATION OF LEAVES OF CALOTROPIS GIGANTEA

Pournima A. Shelar 1 , Sucheta Tikole 2 , Poonam Nalawade 3 And Varsha G. Gharge 3 . 1. Depatment of Pharmacognosy and Phytochemistry, Arvind Gavali College of Pharmacy, Satara, India-415004. 2. Alard college of Pharmacy, Pune, Maharashtra, India. 3. GIPER, Limb, Satara, India-415015. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 22 February 2019 Final Accepted: 24 March 2019 Published: April 2019

Arka (Calotropis gigantea) an important drug of Ayurveda is known in this country from the earliest time. It is commonly known as giant milk weed. It is used for several medication purposes in traditional medicinal system. Most recently Calotropis gigantea is scientifically reported for several medicinal properties viz. the flowers were reported to possess analgesic activity, antimicrobial and cytotoxic activity. Leaves and Arial parts of the plant were reported for anti-diarrhoeal activity, antibacterial activity, antioxidant activity. Roots were reported to contain anti-pyretic activity, cytotoxic activity. The present study is an effort to give detail information regarding macroscopy, microscopy, physical constants, leaf constants, fluorescence analysis and phytochemical screening of leaves extracts of Calotropis gigantea. This study helps in identification of this particular plant species. Also it provides guidelines for identification of plant species.

…………………………………………………………………………………………………….... Introduction:-
The plants posses therapeutic properties or exert Beneficial Pharmacological effects on the animal body are generally designated as "Medicinal Plant". A medicinal plant is any plant which, in one or more of it contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs. 1 Humankind first utilized materials found in environment an empirical basis to cure various ailments. Natural products from plants and animals traditionally have provided the pharmaceutical industry with one of its important sources of lead compounds in search of new drugs and medicines. The search for new pharmacologically active agents from natural resources such as plants, animals and microbes led to discovery of many clinically useful drugs. 2,3 Since the beginning of civilization, human beings have worshiped plants and such plants are conserved as a genetic resource and used as food, fodder, fibre, fertilizer, fuel, febrifuge and in every other way 4 , Calotropis gigantea is one such plant 5 .
Calotropis belong to Asclepidaceae family. It is also known as Akada, Aak, Mandar, Aakh etc. It has two species procera and gigantean. In ancient ayurvedic medicine the plant Calotropis gigantea is known as "Sweta Arka" and Calotropis procera as "Raktha Arka". Both of them are often similar in their botanical aspects and also have similar pharmacological effects. 6

Collection of Plant material
The leaves of Calotropis gigantea were collected in the month of June from Satara district. The leaves were authenticated from Department of Botany. Yashawantroa. Chavan. Institute of Science, Satara, Maharashtra, India. Specimen voucher was deposited in the college herbarium for future reference. The fresh leaves were used for study of macroscopic and microscopic characters. Collected Leaves were shade dried and coarsely powdered. This coarse powder was used for determination of extractive values, ash value, LOD and preliminary phytochemical investigation.

Determination of physical constants Determination of Ash Values: 14 -
Ash values are helpful in determining the quality and purity of a crude drug, especially in the powdered form. The objective of ashing vegetable drugs is to remove all traces of organic matter, which may otherwise interfere in an analytical determination. On incineration, crude drugs normally leave an ash usually consisting of carbonate, phosphates and silicates of sodium, potassium, calcium and magnesium. The total ash of a crude drug reflects the care taken in its preparation. A higher limit of acid insoluble ash is imposed, especially in cases where silica may be present or when the calcium oxalate content of the drug is very high.

Total ash value:
Weigh accurately about 2 to 3 gm of the powdered drug in a tarred silica crucible. Incinerate at a temprature not exceeding 450 0 C for 4 hrs, until free form carbon, cooled and weighed. Calculate the percentage of ash with reference to air dried drug using following formula, % Total ash value = Wt. of total ash x 100 Wt. of crude drug taken 1363 Water soluble ash value: The ash was boiled with 25ml of water, filtered & collected the insoluble matter on an ashes filter paper, washed with hot water and ignited in a tarred crucible at a temperature not exceeding 450 o C for 4 hr. cooled in desiccators, weighed and subtracted the weight of insoluble matter form the total wt. of ash. The difference in weight represented weight of water soluble ash. Calculated the percentage of water soluble ash with reference to the air dried drug using the following formula-% Water soluble ash value = Wt. of total ash -Wt. of water insoluble ash x 100 Wt. of crude drug taken Acid insoluble ash value:-Boil the ash for 5 min with 25 ml of 2 M HCL. Filtered and collected the insoluble matter on an ashless filter paper, washed with hot water & ignited in a tarred crucible at a temp. Not exceeding 450 o C for 4 hr. cooled in desiccators and weighed. Calculate the percentage of acid insoluble ash with reference to the air dried drug using following formala, % Acid insoluble ash value = Wt. of acid insoluble ash value x 100 Wt. of crude drug taken Extractive Values 14 : The extractive values for various solvents of air dried sample were evaluated. Petroleum ether soluble extractive value: 5 gms of air dried powder of Calotropis gigantea, was macerated with 100 ml of pet. Ether in a closed flask, shaking frequently during the first 6 hr and allowed to stand for 18 hr separately. Thereafter, it was filtered rapidly taking precaution against loss of pet. Ether. Evaporated 25 ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 0 C and weighed. Percentage petroleum ether soluble extractive was calculated with reference to the air dried drug.
Chloroform soluble extractive value: 5 gms of air dried powder of Calotropis gigantea, was macerated with 100 ml of chloroform in a closed flask, shaking frequently during the first 6 hr and allowed to stand for 18 hr separately. Thereafter, it was filtered rapidly taking precaution against loss of chloroform. Evaporated 25 ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 0 C and weighed. Percentage chloroform soluble extractive was calculated with reference to the air dried drug.
Methanol soluble extractive value: 5 gms of air dried powder of Calotropis gigantea, was macerated with 100 ml of methanol in a closed flask, shaking frequently during the first 6 hr and allowed to stand for 18 hr separately Thereafter, it was filtered rapidly taking precaution against loss of methanol. Evaporated 25 ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105 0 C and weighed. Percentage methanol soluble extractive was calculated with reference to the air dried drug.
Aqueous soluble extractive value: 5 gms of air dried powder of Calotropis gigantea, was macerated with 50 ml of water at 80 0 C in a closed flask, shaking frequently and allowed to stand for 10 min so as to cool it and filtered. 5 ml of filtrate was transferred to an evaporating dish, which was 7.5 cm in diameter, the solvent was evaporated on water bath, allowed to dry for 30 min. finally dried in an oven for 2 hr at 100 0 C and residue was weighed. Percentage of water soluble extractive was calculated with reference to the air dried drug.

C. Loss of drying 15 :-
Loss on drying is the loss of mass expressed as per cent w/w. The test for loss on drying determine both water and volatile matter in the crude drug. Moisture is an inevitable component of crude drug, which must be eliminated as far as possible.

1364
An accurately weighed quantity of about 5 g of powdered drug was taken in a tarred porcelain dish. The powder was distributed evenly. The porcelain dish kept open in vacuum oven and the sample was dried at temperature 110 0 C for 2 hr until a constant weight was recorded. Then it was cooled in desiccators to R. T., weighed and recorded % Loss of drying was calculated using the following formula, % Loss of drying = Loss in weight of the sample x 100 Wt. of crude drug taken

Fluorescence analysis of Leaf powder
Many drugs shows fluorescence when their powder is exposed to ultraviolet radiation. It is important to observe all materials on reaction with different chemical reagents under U.V. light. The fluorescence characteristics of powdered drug were studied under U.V. light after treating with different chemical reagents is reported. Fluorescence analysis was carried out according to the method of Chase and Pratt 16 and Kokoski. 17 Macroscopic Characteristic 18, 19.
The macroscopy of fresh leaves were studied according to standard methods.

Microscopic characteristics 20
For microscopy hand section of leaf was taken, stained & mounted following usual micro-techniques. 3. Place stage micrometer on the microscope and using 16 mm objective, draw a line equivalent to 1 mm as seen through the microscope. 4. Construct a square on this line. 5. Move the paper so that the square is seen in the eye piece in the center of the field. 1365 6. Place the slide with the cleared leaf (Epidermis on the stage). 7. Trace off the veins which are included within the square, completing the outlines of those islets which overlap two adjacent sides of the square. 8. Count the number of veins islet in the square millimeter. Where the islets are intersected by the sides of the square, include those on two adjacent sides and include those islet on the other sides. 9. Find the average number of vein islet from the four adjoining Squares, to get the value for one sq.mm.

Leaf Constants
Determination of vein let termination number:-Procedure:-1. Clear a piece of the leaf by boiling in chloral hydrate solution for about thirty minutes. 2. Arrange a camera Lucida and drawing board for making drawings to scale. 3. Place stage micrometer on the microscope and using 16 mm objective, draw a line equivalent to 1 mm as seen through the microscope. 4. Construct a square on this line. 5. Count the number of terminations present within the square. 6. Find the average number of vein let termination number from the four adjoining squares to get the value for one sq. mm.

Preparation of Extract 22.
The pulverized dried Calotropis gigantea leaves were extracted with methanol by using soxhlet apparatus. The powder of Calotropis gigantea leaves were also macerated with chloroform water. Methanolic and aqueous leaf extracts were filtered & evaporated to dryness.

Phytochemical Screening 22,23
Preliminary chemical tests for methanolic and aqueous leaf extracts were carried out according to the standard procedures described by Kokate and Horborne.

Chromatoghraphic Studies 24
Thin layer chromatographic studies were carried out for various extracts to confirm the presence of different phytoconstituents in these extract. TLC is a mode of liquid chromatography, in which, the extract is applied as a small spot or band at the origin of thin sorbent layer supported on a glass/ plastic/ metal plates. The mobile phase migrates through the stationary phase by capillary action. The separation of solutes takes place due to their differential absorption/partition coefficient with respect to both mobile and stationary phases. Each separated component has same migration time but different migration distance.
The mobile phase consists of a single solvent or a mixture of solvents. Although a number of sorbent like silica gel, cellulose, polyamide, alumina, chemically modified silica gel etc. are used, silica gel (type 60) is most commonly used sorbent. Handmade plates are prepared by using techniques like pouring, dipping, or spraying. Now days, readymade percolated plates are also available. The plates need to be activated at 110 o C for 1 hr. This removes water/ moisture loosely bound to silica gel surface. The retardation factor (R f ) is calculated using following formula, R f = Distance traveled by sample form base line Distance traveled by solvent form base line

Thin Layer Chromatography 25
Thin layer chromatography is a simple and rapid method carried out using thin layer of absorbent on plates. TLC not only combines the advantage of paper and column chromatography but in certain aspects it is found to be superior to either method. The extract was subjected to thin layer chromatography for the presence of phytoconstituents. In this technique, the silica gel-GF 254 (For TLC) was used as an adsorbent and plates were prepared by spreading technique, then air dried for an overnight and activated for one hour at 110 0 c and used.

Principle-
In the classification of chromatographic methods, TLC has been included under both absorption and partition chromatography, as various materials of different adsorptive power are used in TLC, the separation of components is not always by adsorptive phenomenon. Separation may result due to adsorption or partition or by both phenomenon depending upon the nature of adsorbents used on plates and solvent system used for development.

Preparative thin layer chromatography 26
A thick layer of silica gel GF-254 was coated on the square shaped plate and activated 110 0 for one hour. The broad band (2 mm width) of extracted sample was applied on the plate. The details of plate as follows-Plate dimension: 10x20 cm Adsorbent : silica gel GF 254 Activation : 100C for one hour Band parameter: Width -2mm, Length-8mm The plate was developed in a saturated chamber having desired solvent system. After developing the plate was dried and if the band gives fluorescence then it can be easily scraped. Otherwise a small portion of the band was sprayed with detecting agent, by taking care to avoid the exposure of remaining plate to spray reagent .Then the band is scraped by measuring the height of sprayed band. The scraped band was then suspended in desired solvent and filtered on what man filter paper no-1 and washed several times with same solvent. The filtrates were combined and concentrated and reduced to dryness .This procedure was followed for several scrapings. Then the resulted compound was run with original sample to confirm the isolation and subjected to U.V absorption and I.R. spectroscopy studies for identification and partial chacterization.

IR Isolated Compound 27,28,29
IR Spetrum was recorded in IR -Spectrometer in 400-4000 frequency in cm -1 for isolated steroids and flavonoids. IR spectrum of compound was carried in KBR pellet and the important absorption can be correlated.

Result:-Collection and Authentication of leaves of Calotropis gigantean
The leaves of Calotropis gigantea were collected and authenticated. These leaves were subjected to further evaluation.

Determination of Physical constants:-
The Ash Values (Total Ash, Acid insoluble ash, and Water soluble ash), Extractive values (Pet. Ether, Chloroform, Methanol and Water soluble extractives) of leaf powder are given in table-1.

Midrib: Upper epidermis and Lower epidermis:
Transverse sections through the midrib showed an upper and lower, single-layered epidermis that was externally covered with a thick, striated cuticle, a few epidermal cells on both lower and upper surfaces, parenchymatous cells that were thin-walled and isodiametric to circular. Intracellular spaces were present in ground tissue. The upper and lower epidermis was covered externally with a thick, striated cuticles. Lower epidermis is similar to upper epidermis.

Collenchyma:
Collenchyma present below upper and above lower epidermis, containing groups of cellulosic cells. These are thick walled cellulosic cells, present above the lower epidermis.

Vascular bundle:
Two vascular bundles are arranged in ring and composed of bicollateral and open vascular bundles. The xylem consists mostly of vessels and tracheids.

Mesophyll:
The mesophyll was seen to be differentiated into a palisade and spongy tissues, 1368

Palisade:
Below the upper epidermis were three rows of elongated, closely arranged, palisade parenchyma are present.

IR of Isolated Compound
Spetrum was recorded in IR -Spectrometer in 400-4000 frequency in cm -1 for isolated compound.