*2.1.1 Collection of plant materials for authentication and for anatomical studies*

The collected fruit of *Calotropis procera* from Titupathi, at the area of nalla malai forest, India. *C. procera* collected in the month of December, 2018. The *C. procera* fruit was authenticated by Prof. K. Madhavachetty, Department of botany, Srivenkateswara University, Andhra Pradesh, India. The given voucher for specimen number is 1221, deposited at College of Pharmaceutical Sciences, Andhra University.

#### *2.1.2 Collection of specimens*

The collected plant species are too healthy for the proposed study, specimens of organs are cutted and soaked into 70% of 90 ml ethyl alcohol, 5 ml of Formalin and 5 ml of acetic acid [2]. The organs of specimens are dehydrated with graded series of tertiary butyl alcohol as per the guidelines given by sass, 1940. Specimens of infiltration were carried by the addition of 58–60°C paraffin wax until TBA solution attained super saturation.

#### *2.1.3 Morphological features*

These plant species grow in all types of soil, collected fresh fruit from 2.8 mts height of the plant early in the morning at 6 am from red loamy soil land. The fruit is 10 cm long and 6 cm width outer and inner layer are green in color and in every fruit contains a plenty of brown-colored seeds more over every seed length is 5.5 to 6.0 mm length and 3.7 mm width many of white silky hairs are present and each silky hair length is 40 to 50 mm in long. Fruit color of the *Calotropis procera* is green in color, strong pungent odor, taste is characteristic/bitter and gummy in nature. Soluble in benzene, ethyl acetate, ethanol, methanol and hydroalcoholic.

#### *2.1.4 Sectioning*

The specimens of paraffin-embedded were sectioned with the help of a rotary microtome, the thickness of each section was 10–12 μm [3]. As per the standard method the obtained sections were stained in to toluidine blue [4] because toluidine blue is a polychromatic stain. The obtained strains are good because some phytochemical reactions were obtained. The obtained dye changed pink color to mucilage blue for the protein bodies, other sections are stained with safranin, fast green and iodine [5].

#### *Detailed Pharmacognostical Standardization Studies on* Calotrophis Procera *(Aiton) Dryand… DOI: http://dx.doi.org/10.5772/intechopen.104549*

For studying the stomatal number, the pattern of venation and distribution of trichomes, sections of paradermal (sections taken parallel to the surface of leaf) as well as clearing of the leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey, s maceration fluid [5] were prepared. Glycerine mounted temporary preparations were made for macerated /cleared materials. Different parts of powdered materials were cleared with NaOH and mounted in glycerine medium after staining. Measurement of different cell components was studied.

#### *2.1.5 Photomicrographs*

Different magnifications of photographs are taken with the help of Nikon labphoto 2 microscopic unit, bright fields were used for normal observation. The polarized light was used for the identification of Different kinds of tissues, crystals, stone cells, starch grains, fibers, lignified and non-lignified cells. Since these obtained structures have fair finger print property, under polarized light they appear bright against the dark background. Magnifications of the figures are indicated by the scale –bars. Descriptive terms of the anatomical features are given in the standard anatomy books [6].

#### *2.1.6 Instrumentation and technique*

The obtained extracts and powder had examined under UV-light (254 nm, 354 nm) [7, 8]. The total ash value, Determination of Acid Insoluble Ash, Determination of Alcohol Soluble Extractive value, and Determination of Water-Soluble Extractive values in the obtained samples were performed, and in Loss on drying measured the amount of water and volatile oils, Swelling index, Foaming index were performed according to the standard procedure, for Extractive values, 2gms of an air-dried coarse powder of drug macerated with 40 ml of solvents (Hexane, Ethyl acetate, Chloroform, Acetone, Methanol and Distilled water) in a glass stoppered conical flask with frequent shaking for 6 hrs and then allowed to stand for 2 days, then filtered carefully, taking care against loss of solvent. The filtrate was evaporated in a silica crucible to dryness in the water bath and then dried 105° for 6 hrs, cooled in a desiccator for 30 min and weighed without delay [9]. Fluorescence Analysis of the drug was observed in UV light using various extracts of the drug. The drug powder was treated separately with different solutions [10, 11] and for Preliminary phytochemical studies. The prepared extracts were tested for the different types of chemical constituents present by known qualitative tests. The following tests were carried out on the extracts to detect various phytoconstituents present in them [12–14] were performed according to the official methods described in the Indian pharmacopeia (1996) and WHO guidelines on quality control methods -for medicinal plant materials.
