Phytochemical Investigation of Rosa bourboniana: A Spectroscopic Study for the Identification of ?-sitosterol

The bond between humans and plants has been present since the dawn of humanity, and recently, with the rising global demand for medicinal plant derivatives, this relationship has intensified.(Charlady, et al., 1999)  The relationship between the humans and plants has existed since existence of human beings and curtly due to the worldwide rise in the need for botanical derivatives medicines the relationship has become stronger.(Newman, et al,2000),Plants contain a diverse combination of various chemical compounds that can differ significantly due to several factors such as lants possess a complex blend of diverse chemical components that can differ significantly.(Kaur,etal.,2009) This natural variability in the chemistry might negatively impact the effectiveness of medicinal plants. Thus, in spite of usage of plant derived product constantly as therapeutic agent it is the necessity of scientist ensure the quality of the herbal drugs and formulation. (Butler MS.2004)

MATERIAL AND METHODS:

All chemicals are collected from analytical grade.

Collection and Preparation of Plant Extract:

In vitro test includes the phytochemical screening test. Rosa Bourbonic disport was collected from botanical garden and authenticated. Then the hips were washed toughly with the tap water, shade dried in 6-7 day in in room with room temperature good airflow and were not expose to direct sunlight. crushed to fine powder and store in air tight container. Preparation of Extraction: Method was used and methanol extract preparation of plant. To obtain an methanolic extract of RBD 100 gm of powder was extracted in 70% methanol by maceration method. Macerated by using maceration tube covered with aluminium foil and cotton in it. This simplicial methanol mixture was stirrer every day twice a day both in morning and evening without touching the cotton. after that extract was evaporated by using rotatory evaporator.

Phytochemical Testing:

Phytochemical analysis was performed on leaf extracts with various solvents to detect the primary natural chemical groups including tannins, saponins, flavonoids, phenols, terpenoids, alkaloids, glycosides, and steroids. Overall response in these analyses indicates whether a compound is present or absent in the tested extract.

Physiochemical Constants:

Physiochemical constant such as the total Ash value acid insoluble and water insoluble, moisture content and extractive value. (Jofre, et al.,2017)

Determination of Total Ash Value:

grams of the powdered sample were precisely weighed individually in a silicon crucible that had been lit and weighed (at 350°C for an hour). A thin coating of dried leaf material was applied to the crucible's bottom. The crucible was burned in a muffle furnace at a temperature of no more than 450°C until it turned white, signifying that it was carbon-free. After cooling, the crucible was weighed. The air-dried powder was used to compute the percentage of total ash. (Usha, et al.,1984)      

Determination of Acid Insoluble Ash Value:

25 ml of 2 N HCL were used to boil the ash that was produced in accordance with the procedure for calculating total ash for five minutes. After being collected on ash-less filter paper, the insoluble ash was cleaned with hot water. After being moved to a silica crucible that had been previously weighed, the insoluble ash was fired for 15 minutes at a temperature of no more than 450°C. The air-dried powder was used to calculate the proportion of acid-insoluble ash.                                           

Determination of Water-Soluble Ash Value:

The ash used to calculate the total amount of ash was heated with 25 ml of water for five minutes. Once gathered on ash-free filter paper, the insoluble substance was rinsed with hot water after being moved into a silica crucible that had been previously weighed, the insoluble ash was fired for 15 minutes at a temperature of no more than 450°C. To obtain the constant weight, the process was repeated. The weight of the whole amount of ash was deducted from the weight of the insoluble substance. The weight differential constituted the water-soluble ash. The air-dried powder provided the material for water-soluble ash proportion analysis.

Determination of Heavy Metal:

Atomic absorption spectroscopy serves as a prevalent and trusted analytical technique for measuring metals and metalloids in environmental samples. An Atomic Absorption Spectrometry (AAS) Model AA 240 Series instrument was employed to measure the total heavy metal content in the sample. To quantify the amounts of metallic contaminants in the test material, specifically lead, cadmium, arsenic, and mercury. The test sample was digested with 1mol/L HCl to measure arsenic and mercury concentrations. Correspondingly, sample digestion was performed using 1mol/L HNO3 to quantify lead and cadmium content. (Rajeev, et al., 2015)                      

TLC:

A pencil line was traced 2 cm from the bottom of one end on a precoated TLC silica gel plate. The compounds were dissolved in a small amount of ethyl acetate and applied to the marked line on the plate using a capillary tube, then left to dry. The dried plate was put into TLC chamber 1:3 having an ethanol: hexane mixture ratio, and the chamber was sealed. The solvent was set upon the plate by capillary action, when the solvent from front was just about 2cm to upper end of the plate, the plate was removed and a line was drawn to mark the position of the solvent front. Following plate drying, spot development was achieved by spraying 5% sulphuric acid as the reagent. A meter rule was used to measure the Rf values of the spots. (Manikantan, et al.,2017)

Description of Isolated B-Sitosterol Fractions:

Description of isolated B-sitosterol fractions 8,9: UV Visible spectroscopy: Spectrophotometric instruments facilitate the examination of photon spectroscopy across the UV-VIS region. The colour of the utilized compound has a direct impact on absorption in the visible range. The isolated compound CT- 1 sitosterol is white powder with a molecular weight of 414.7g/mol and a melting point of 136O C which corresponds to the molecular formula C19H50O.The UV λ max value of compound B-sitosterol was 216 nm. (Sribatsa et al.,2022)

FTIR:

FTIR has demonstrated itself as an extremely valuable technique for characterizing and determining the chemicals or functional groups (chemical bonds) found in plant extract combinations. It is a fast, non-damaging technique that needs minimal sample preparation. The presence of bands in the infrared spectrum makes possible the qualitative identification of chemical molecules. (Padmasri, et al.,2011)

RESULT:

Table I, II, III, IV show the result of macroscopic appearance, Solubility profile and physiochemically properties, heavy metal analysis.

Macroscopic Appearance

TABLE I

Solubility Profile

TABLE II

Physiochemical properties

TABLE III

HEAVY METAL ANALYSIS:

TABLE IV

IR:

V max: The absorption spectrum showed absorption peaks at 3328.7 cm-1(O-H starching)2922 cm-1and 2102cm-1(aliphatic-H starching),1625 cm -1(C=C absorption peak) and other picks include 1341cm-1(CH2),1006 cm-1(cycloalkane) and 760cm.

Fig.no.1 FTIR -Spectra

Fig.no.2 UV