Quantitative Analysis of Biochemical Constituents in Tulsi (Ocimum sanctum L.)

 Introduction

Tulsi (Ocimum sanctum L.), commonly known as Holy Basil, is one of the most important medicinal plants in India. The name Tulsi is derived from the Sanskrit word meaning “the matchless one” or “the incomparable,” reflecting its esteemed status in traditional healthcare systems [1]. Besides its prominent role in Ayurveda, Tulsi has also been used in the Greek, Roman, and Unani systems of medicine for its wide range of therapeutic properties [2]. Tulsi is a perennial aromatic herb belonging to the family Lamiaceae and is widely cultivated in tropical and subtropical regions [3]. The plant grows erect with several branches and attains a height of approximately 30–60 cm at maturity. Its leaves are simple, opposite, elliptic to oblong in shape, with entire or slightly serrated margins. The leaves possess a characteristic aroma due to the presence of essential oils rich in bioactive compounds [4]. The flowers are small, purplish in color, and borne on elongated racemes, while the fruits contain small reddish-yellow seeds [3]. Medicinal plants serve as a valuable source of bioactive compounds used in healthcare and drug development. Among them, species of the genus Ocimum are particularly important because of their antioxidant, antimicrobial, anti-inflammatory, antidiabetic, and immunomodulatory activities [1]. Ocimum sanctum mainly occurs in two varieties: Krishna Tulsi, characterised by purple leaves, and Rama Tulsi, which possesses green leaves [3]. Owing to its rich phytochemical composition and therapeutic potential, Tulsi

continues to be extensively studied for its medicinal and pharmaceutical applications [2].

Methodology

The present study was conducted on the campus of Starex University, Binola Village, Gurugram District, Haryana, India. Plant material

Fresh leaves of Tulsi (Ocimum tenuiflorum L.) were used for the biochemical analysis.

Selection and Collection of Leaf Samples

Healthy, mature leaves free from disease, insect damage, and discoloration were selected to ensure uniformity and reliability of results. Leaves were collected from plants of the same variety to minimize variation. Sampling was carried out during the early morning hours when metabolic activity and moisture content were relatively stable. The leaves were harvested using clean hands or sterilized scissors and immediately transferred to clean polythene bags for transportation to the laboratory. Wet leaves and leaves collected immediately after rainfall were avoided to prevent interference with biochemical estimations.

Biochemical Analysis

The collected leaf samples were analyzed under laboratory conditions for the estimation of important biochemical constituents. The parameters studied included:

• Chlorophyll (a, b, and total chlorophyll)
• Total sugars
• Reducing sugars
• Total carbohydrates
• Lipids

 Carotenoids Alkaloids Phenols

Standard biochemical methods were employed to determine the concentration of these constituents and to evaluate the nutritional and medicinal significance of Tulsi leaves.

MATERIAL REQUIRED

INSTRUMENTS: Test tube, Conical flask, Beaker, Measuring cylinder, Cuvette, Lab dropper, Spatula, Conical flask, Filter paper, Hand gloves, Face mask, Mortar and pestle

Chemicals Required: Distilled water, Calcium carbonate, Petroleum ether, Sulphuric acid, 80% Acetone, 80% Ethanol, Methanol, Gallic acid, Glucose solution, Acetic acid, Benedict’s reagent, Anthrone Reagent

Biochemical estimation methods:

1.  Estimation of Chlorophyll

Chlorophyll content was estimated by extracting fresh Tulsi leaves (0.5 g) with 80% acetone in the presence of calcium carbonate [5]. The extract was centrifuged, and the supernatant was collected and made up to a known volume. Absorbance was measured at 663 nm and 645 nm using a spectrophotometer. Chlorophyll a, chlorophyll b, and total chlorophyll contents were calculated using standard equations.

2.  Estimation of Carotenoids

Carotenoids were extracted from fresh Tulsi leaves using 80% acetone [5]. The extract was centrifuged to obtain a clear supernatant, and absorbance was recorded at 480 nm, 663 nm, and 645 nm. The carotenoid content was calculated using standard spectrophotometric formulae.

3.  Estimation of Total Lipids

Total lipids were estimated by solvent extraction. Dried Tulsi leaf powder (0.5 g) was treated with petroleum ether and heated gently to facilitate lipid extraction [6] The extract was filtered, and the solvent was evaporated on a water bath. The residue obtained after evaporation represented the lipid content of the sample.

4.  Estimation of Total Sugars

Total sugar content was determined using Benedict’s reagent [7]. Fresh leaf extract was prepared by grinding leaves in distilled water followed by centrifugation. The extract was mixed with Benedict’s reagent and heated in a boiling water bath. The development of color from blue to red indicated the presence of sugars, and the intensity of the color corresponded to the sugar concentration.

5.  Estimation of Total Carbohydrates

Total carbohydrate content was estimated by the Anthrone method [8]. Fresh leaf extract was reacted with Anthrone reagent in the presence of concentrated sulphuric acid and heated in a water bath. A green-colored complex was formed, and its absorbance was measured spectrophotometrically. The carbohydrate concentration was determined using a glucose standard curve.

6.  Estimation of Total Phenols

Total phenolic content was estimated using the Folin–Ciocalteu method [8]. Methanolic leaf extract was reacted with Folin–Ciocalteu reagent and sodium carbonate solution. After incubation, a blue-colored complex developed, and absorbance was measured at 765 nm using a spectrophotometer. The phenolic content was quantified using gallic acid as the standard.

7.  Estimation of Alkaloids

Alkaloid content was estimated by extracting dried Tulsi leaf powder with acidic ethanol [8]. The extract was filtered and concentrated, followed by precipitation of alkaloids using ammonium hydroxide. The precipitate was collected, dried, and weighed. The alkaloid content was expressed as a percentage of the dry weight of the sample.

8.  Estimation of Reducing Sugars

Reducing sugar content was estimated using the 3,5-Dinitrosalicylic Acid (DNS) method [7]. Fresh Tulsi leaves (1 g) were washed, homogenized with distilled water, and filtered to obtain a clear extract. The extract was reacted with DNS reagent and heated in a boiling water bath, resulting in the formation of an orange-red colored complex. The intensity of the color, measured spectrophotometrically at 540 nm, was directly proportional to the concentration of reducing sugars present in the sample. Glucose was used as the standard for quantification.

Result:

1. Chlorophyll content–

The chlorophyll pigment of the Tulsi leaf sample was estimated spectrophotometrically. The chlorophyll a [0.121 mg/g FW] was higher than chlorophyll b [ 0.060 mg/g FW], while the total chlorophyll content was [0.181 mg/g FW]. The higher amount of chlorophyll a indicates that it is the primary photosynthetic pigment responsible for light absorption and energy conversion. Chlorophyll b acts as an accessory pigment

• Carotenoids content –

The carotenoid content [ 0.0216 mg/g FW] was also observed in the sample. Carotenoids are important accessory pigments that protect chlorophyll molecules from oxidative damage and help in light harvesting. Their presence suggests that the sample possesses protective antioxidant mechanisms.

Absorbance 480 – 2.014

Absorbance 663 – 2.457

Absorbance 645 – 1.900

3. Carbohydrate content-

Carbohydrates are the major products of photosynthesis and serve as the primary source of energy in living organisms. In the present study, the total carbohydrate content was found to be

0.8 g/g, indicating that the sample contains a high amount of carbohydrates. This high carbohydrate content may contribute to the nutritional importance of the plant sample. Carbohydrates also plays a structural role in plant cells as components of cellulose and other polysaccharides.

Measurement – the absorbance of all tubes was recorded at 620 nm using a spectrophotometer

• Blank absorbance ₌ 0.000

4] Phenol content –

The total phenol content was estimated as 2.24 mg/g. Phenolic compounds are secondary metabolites widely known for their antioxidant, antimicrobial, anti –inflammatory, and therapeutic properties. The presence of appreciable phenol content in the sample indicates potential medicinal value. Phenol help plant resist microbial attract and environmental stress.

5] Reducing sugar –

Reducing sugars are simple sugar capable of acting as reducing agents due to the presence of free aldehyde or ketone groups. They include glucose, fructose, lactose and maltose. In the present investigation, the reducing sugar content was estimated to be 6.8 mg /g

During qualitative analysis, the development of green color indicated the presence of reducing sugar in low to moderate quantity.

Reading –

 Sample absorbance = 0.43

6] Alkaloid content –

Qualitative analysis of alkaloids in the Tulsi leaf extract was carried out using Mayer’s test, Wagner’s test, dragendorffs test. The observation confirmed the presence of alkaloids in sample.

7]  Estimation the total sugar

The total sugar content of Tulsi leaf was analyzed by qualitative biochemical test. During the experiment, the development of green color was observed in the reaction mixture. The appearance of green color indicates the presence of a low concentration of total sugar in the leaf sample.

This result confirms that Tulsi leaf contains soluble sugar, but in comparatively small amounts. Sugar are important primary metabolites that serve as an immediate source of energy and are involved in various physiological processes such as respiration, growth, biosynthesis of other organic compounds.

The green color indicates the presence of a low amount of sugar in a sample

8]  Lipid content –

The lipid content was 0.01 mg /g FW which was comparatively low. This suggests that the sample is not a major source of fats but may still contain essential fatty substance required for membrane structure and metabolism.

 

References

1. Prakash, P., & Gupta, N. (2005). Therapeutic uses of Ocimum sanctum Linn. (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian Journal of Physiology and Pharmacology, 49(2), 125–131.
2. Pattanayak, P., Behera, P., Das, D., & Panda, S. K. (2010). Ocimum sanctum Linn. A reservoir plant for therapeutic applications: An overview. Pharmacognosy Reviews, 4(7), 95–105. https://doi.org/10.4103/0973-7847.65323
3. Warrier, P. K. (1995). Indian Medicinal Plants: A Compendium of 500 Species (Vol. 4). Orient Longman.
4. Kothari, S. K., Bhattacharya, A. K., & Ramesh, S. (2004). Essential oil yield and quality of methyl eugenol-rich Ocimum tenuiflorum L. grown in South India as influenced by method of harvest. Journal of Chromatography A, 1054(1–2), 67–72. https://doi.org/10.1016/S0021-9673(04)01467-0

5. Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. In L. Packer & R. Douce (Eds.), Methods in Enzymology (Vol. 148, pp. 350–382). Academic Press. https://doi.org/10.1016/0076-6879(87)48036-1

• Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. https://doi.org/10.1139/o59-099.

7. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428. https://doi.org/10.1021/ac60147a030

Sadasivam, S., & Manickam, A. (2008).Biochemical Methods (3rd ed.). New Age International (P) Limited. ISBN: 9788122421408.