Introduction

Catharanthus roseus (L.) G. Don, commonly known as Sadabahar or Madagascar periwinkle, is a perennial herb belonging to the family Apocynaceae. It is widely cultivated as an ornamental plant and is of great medicinal importance due to the presence of alkaloids such as vincristine and vinblastine. The plant exhibits typical dicotyledonous morphological features with well-developed vegetative and reproductive organs. The presence of numerous bioactive alkaloids and their pharmaceutical significance[1]. The biosynthesis of these alkaloids and emphasized the plant’s medicinal value[2]. The significant changes in chlorophyll, proteins, carbohydrates, and antioxidant enzymes under different environmental conditions[3].

In addition, the plant exhibits antioxidant, antimicrobial, antidiabetic, and anti-inflammatory activities. Biochemical estimation of different parameters such as chlorophyll, carotenoids, proteins, carbohydrates, phenols, flavonoids, and antioxidant enzymes provides important insights into its physiological status, metabolic processes, and medicinal potential[4].

The analysis of these biochemical constituents helps in understanding plant growth, development, stress responses, and the accumulation of therapeutically important compounds. Therefore, the present study focuses on the biochemical estimation of various parameters in Catharanthus roseus to evaluate its physiological and medicinal significance and to contribute to its pharmaceutical and agricultural applications.

Materials and Methods Preparation of plant extract:

Fresh and healthy leaves of Catharanthus roseus (L.) G. Don were collected from the campus of Starex University, Bhora Kalan. The plant were identified based on standard taxonomic keys. The collected samples were washed thoroughly with distilled water to remove dust and contaminants.

The leaves were shade-dried at room temperature for several days and then ground into fine powder using a mortar and pestle. The powdered samples were stored in airtight containers for further biochemical analysis. About 1 g of dried leaf powder was taken and homogenised with 10 ml of appropriate solvent (such as ethanol, methanol, or distilled water, depending on the test). The homogenate were centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. This extract were used for the estimation of various biochemical parameters.

Materials Required: Test tube, Cuvette, Filter paper, Conical flask, Lab dropper, Hand Gloves, Beaker, Spatula, Measuring cylinder, Conical funnel, Mortar and Pestle, UV-Visible spectrophotometer, Centrifuge Machine, Weighing Balance, Heating Mantle, Water bath, Hot air oven

Chemicals Required: 80%Acetone, Aluminium Chloride, Benedict’s Reagent, Distilled Water, Methanol, Anthrone Reagent, Calcium Carbonate, Sodium Carbonate, Dragendorff’s Reagent, Petroleum Ether, Gallic Acid (Standard), Quercetin Hydrate 95%, Conc. Sulphuric acid, Acetic Acid, Potassium Acetate, 80% Ethanol, Glucose Standard Solution, Folin & Ciocalteu’s Phenol Reagent.

Biochemical Parameters Estimation:

Chlorophyll Estimation:

Chlorophyll pigments are extracted with 0.5g of fresh leaf in 80% acetone, add a pinch of calcium carbonate and quantified spectrophotometrically at 645 nm (Chl b) and 663 nm (Chl a). Total chlorophyll calculated using Arnon’s equations based on specific absorbance coefficients.

Total Chlorophyll (mg/g) = 20.2 (A663) + 8.02 (A645) × Total volume of extract

1000 × Sample weight (g)

Carotenoid Estimation:

Carotenoids are accessory pigments present in plant leaves[5]. They are extracted using organic solvents like acetone, and their absorbance is measured at 480 nm and 510 nm. The carotenoid content is calculated using standard equations based on absorbance values.

Lipid Estimation:

Lipids are non-polar compounds that are soluble in organic solvents such as petroleum ether. Fresh leaves of Catharanthus roseus were used for lipid extraction. The sample was ground

and treated with petroleum ether, resulting in a light yellow extract, indicating the presence of lipids[6]. After filtration/centrifugation, a clear extract was obtained. Upon evaporation of petroleum ether, a greasy residue remained, confirming lipid extraction.

Reducing Sugar Estimation:

Benedict’s reagent contains copper sulfate, sodium carbonate, and sodium citrate. Reducing sugars (such as glucose, fructose) reduce Cu²⁺ ions to Cu⁺ ions under alkaline conditions. This forms cuprous oxide (Cu₂O), which appears as a coloured precipitate. After adding Benedict’s reagent to the leaf extract of Catharanthus roseus and heating it in a boiling water bath, the solution turned from blue to green with slight turbidity.

The appearance of green colour indicating low concentration of reducing sugars in the leaf extract of Catharanthus roseus.

Total Sugar Estimation:

Total sugars were estimated by the Anthrone method, based on acid hydrolysis and dehydration of carbohydrates to form furfural derivatives, which react with Anthrone reagent to produce a stable green-colored complex. The appearance of green colour indicates the presence of total sugars (both reducing and non-reducing sugars) in the leaf extract of Catharanthus roseus[7].

Carbohydrate Estimation:

Fresh leaves of Catharanthus roseus were collected, washed thoroughly, and blot-dried before weighing 1g of the sample. The leaf material was homogenised in distilled water and centrifuged at 5000 rpm for 10 minutes to obtain a clear supernatant. Total carbohydrate content was determined by the Anthrone method[8]. A glucose stock solution (1 mg/ml) was used to prepare a series of standard concentrations. Both standard solutions and sample extracts were treated with Anthrone reagent and heated in a boiling water bath, leading to the formation of a bluish-green coloured complex. The intensity of the colour developed was measured spectrophotometrically at 620 nm. A standard calibration curve was plotted using glucose standards, and the carbohydrate content of the sample was calculated from this curve. Carbohydrate (mg/g) = Amount from graph × Total volume

Sample weight×Aliquot

Total Phenol Content Estimation:

Total phenolic content in Catharanthus roseus leaves was determined using the Folin–Ciocalteu method. The extract was treated with Folin–Ciocalteu reagent and sodium carbonate to develop a blue-colored complex, and absorbance was measured at 765 nm using a spectrophotometer. Gallic acid was used as the standard for calibration by preparing a series of known concentrations to construct a standard curve. The results were expressed as mg of gallic acid equivalents (GAE) per gram of fresh weight[9].

Total phenol (mg/g) = Concentration from standard curves× volume of sample

Sample weight

Alkaloids Estimation:

Total alkaloids in Catharanthus roseus were detected using Dragendorff’s reagent, which produces an orange-brown precipitate with alkaloids. The formation of precipitate confirms the presence of alkaloids[10].

Flavonoids Estimation:

Total flavonoid content in Catharanthus roseus leaves was determined using the aluminium chloride colourimetric method. The prepared plant extract was treated with aluminium chloride reagent, resulting in the formation of a yellow-coloured flavonoid–aluminium complex. After proper incubation, the intensity of the developed colour was measured spectrophotometrically at 415 nm. A standard curve was constructed using quercetin as the reference compound, and the flavonoid content of the sample was calculated accordingly. The quercetin standard solutions showed a gradual increase in absorbance with increasing concentration, indicating a linear relationship.

The spectrophotometric analysis of the sample showed an absorbance value of 0.649 at 645nm (A645) and 1.201 at 663nm (A663), which were utilised for the estimation of chlorophyll ‘a’, chlorophyll ‘b’, and total chlorophyll content in Catharanthus roseus.

Carotenoids Contents:

Image 2. Spectrophotometer absorbance of the sample at 480nm &510nm for carotenoid estimation.

The total carotenoid content of Catharanthus roseus was 0.324 mg/g of fresh weight, indicating the presence of accessory photosynthetic pigments in moderate amounts.

Lipids Contents:

Image 3(a). Lipid extraction from Catharanthus roseus using petroleum ether, showing lipid residue.

The lipid content of Catharanthus roseus was estimated by solvent extraction using petroleum ether. The extraction yielded 0.06g of lipid residue from 0.5g of sample, resulting in a total lipid content of 12% (w/w).

Image 3(b). Lipid extraction from C.roseus using petroleum ether, showing lipid residue.

Reducing Sugar Contents:

Image 4. Reducing sugar test of Catharanthus roseus showing green colouration, indicating low sugar content.

The qualitative estimation of reducing sugars in Catharanthus roseus using Benedict’s reagent showed a green colour, which confirms the presence of reducing sugars in low amounts. This indicated a partial reduction of Cu2+ ions to Cu+ due to a small quantity of reducing sugars in the sample.

Total Sugar Contents:

Image 5. Total sugar showing green colour in Catharanthus roseus leaf extract.

The total sugar content in Catharanthus roseus leaf extract was determined to be 1.8mg/g fresh weight using spectrophotometric analysis. The development of green colour during the reaction confirmed the presence of total sugar in the sample.

Total Carbohydrates Contents:

Image 6. Estimation of carbohydrate showing moderate intensity in plants.

Spectrophotometric estimation of carbohydrate content of Catharanthus roseus leaf extract showing a value of 1.64mg/g fresh weight at 620nm, indicating a moderately high level with characteristic green colour development.

Table 1. Standard reading of Carbohydrates :

Phenol Contents:

Image 7. Spectrophotometric estimation of total phenolic content at 765nm.

The total phenolic content was quantitatively estimated as 5.46mg/g fresh weight using spectrophotometric analysis at 765nm, confirming the appreciable accumulation of phenolic compounds in the sample.

Alkaloids Contents:

The presence of alkaloids was confirmed by the formation of a strong dark brown precipitate, indicating a high concentration of alkaloid compounds in the sample.

Flavonoids Contents:

The concentration was calculated using standard calibration curve plotted between absorbance and concentration.

The concentration was calculated using standard calibration curve plotted between absorbance and concentration.

Conclusion:

The biochemical estimation of different parameters in Catharanthus roseus demonstrated the presence and relative concentration of important biomolecules such as chlorophyll, proteins, carbohydrates, phenolic compounds, and other primary and secondary metabolites. These biochemical constituents play vital roles in the plant’s growth, metabolism, and defense mechanisms. The results indicate that Catharanthus roseus possesses significant biochemical richness, which supports its well-known medicinal value and its use in pharmaceutical applications. The study also highlights that variations in these biochemical parameters may occur due to environmental conditions, plant age, and physiological status. Overall, the biochemical analysis provides valuable information on the metabolic profile of Catharanthus roseus and emphasizes its importance as a medicinal plant with potential therapeutic and research applications.

References:

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