Gas Chromatographic Profiling and Characterization of Essential Oils from Ocimum tenuiflorum

        

  Gautam P. Vadnere*, Md. Rageeb Md. Usman, Shreya C. Jain, Swati Turkhade, Anuja Patil, Karuna Patil

Smt. Sharadchandrika Suresh Patil College of Pharmacy, Chopda, Maharashtra, India

*Correspondence: rageebshaikh@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4501  

Article Information		Abstract
Review Article Received: 07/05/2025 Accepted: 12/05/2025 Published: 31/05/2025   Keywords Ocimum tenuiflorum; Essential oil; Steam distillation; GC-FID; Eugenol; Phytochemical profiling		Ocimum tenuiflorum (Holy Basil) is an important medicinal herb extensively employed across traditional healing systems like Ayurveda, Siddha, and Unani due to its wide range of therapeutic benefits. In the present research, essential oils were obtained from the entire plant including leaves, stems, flowers, roots, and seeds through steam distillation. The extracted oil underwent detailed evaluation using qualitative and quantitative phytochemical screening, physicochemical tests, and gas chromatography with flame ionization detection (GC-FID). Preliminary phytochemical tests confirmed the presence of key constituents such as alkaloids, flavonoids, phenolics, terpenoids, and glycosides. Quantitative analysis revealed total phenolic content of 1.61%, flavonoids at 1.56%, and alkaloids at 0.91%. GC-FID analysis identified 24 chemical constituents in the essential oil, with eugenol being predominant (83.08%), followed by citronellol, sabinene, and carvacrol. These compounds are known to exhibit significant antimicrobial, antioxidant, and anti-inflammatory properties. This study supports the pharmacological relevance of O. tenuiflorum and offers a scientific basis for its traditional applications and further exploration in pharmaceutical industries.

INTRODUCTION

Since ancient times, Ocimum tenuiflorum (synonym: Ocimum sanctum), commonly referred to as Tulsi and belonging to the family Lamiaceae (Labiatae), has held a sacred status in India and is widely recognized for its extensive medicinal properties and disease-preventive benefits. Known in English as Holy Basil, this revered herb originates from India and is now widely cultivated and distributed across the globe [1]. Historical references to its medicinal use can be traced back to the Rigveda (circa 3500–1600 B.C.), highlighting its role as one of the earliest known therapeutic plants in Indian tradition [2]. The plant is favored in traditional medicine not only due to its accessibility and cost-effectiveness but also for its safety and efficacy. The entire plant—including leaves, stem, flowers, roots, and seeds—has been incorporated into various ancient medical systems such as Ayurveda, Siddha, Greek, Roman, and Unani, due to its diverse pharmacological actions. Documented therapeutic benefits of O. tenuiflorum include analgesic, anticancer, antiasthmatic, antiemetic, diaphoretic, antidiabetic, antifertility, hepatoprotective, hypotensive, hypolipidemic, and adaptogenic (antistress) effects [3,5].

 

Figure 1: Ocimum tenuiflorum

The different parts of Ocimum tenuiflorum contain different types of Constituents in varying amounts. The leaves contain a high content Of essential oils which include Toluene,, Camphene, Octane, Benzene, Citronellel, Sabinene, Limonene, Ledol, Dimethylbenzene, Ethyl-2-Methylbutyrate, Eugenol, Terpiniolene, β-elemene, Isocaryophyllene, Iso-eugenol, α- amorphene, α-guaiene, α-humulene, α-terpeneol, Borneol, Calamine, Nerolidol, Carvacrol, Geraneol, Humulene Oxide, Elemol, Tetradecanal, (EZ)-famesol, Cissesquisainenehydrate, Αbisbolol, Selin-11-en-4-α-ol,α-murolene, 14-hydroxy-α-humulene[7,8]. To separate constituents’ extraction is performed in many ways.

 

Figure 2: Structures of chemical constituents [8]

 

Scientific Classification of O. Tenuiflorum

     Table 1: Scientific Classification of O. tenuiflorum [9]

 

MATERIALS AND METHODS

Materials needed:

1)   Fresh or dried tulsi leaves

2)   Distillation apparatus (steam distillation unit)

3)   Water (preferably distilled water)

4)   Condenser

5)   Receiving flask

6)   Separation funnel (optional)

7)   Ice (optional for cooling)

8)   Collection bottles (for storing the essential oil)

Collection of plant material: Leaves of Ocimum tenuiflorum L. (tulsi) were collected from Dhanvantari Garden of Smt. S. S .P. COP, Chopda, Jalgaon, washed with sterile water and dried in shades. Then the samples were powered in mechanical grinder [10].

Authentication of Ocimum tenuiflorum

We specified the genus and species of the Tulsi plant as Ocimum teniuflorum on basis of its botanical classification. Scientific research and herbarium records validate the name Ocimum tenuiflorum. For that we create herbarium of palmorosa plant and authenticate it from botanist. [At Dadasaheb Suresh Patil Science Art Commerce College, Chopda.]

Extraction of Tulsi oil

The extraction of tulsi oil (essential oil from Ocimum tenuiflorum, commonly known as holy basil or tulsi) is typically done using steam distillation, as it is the most effective method for obtaining essential oils from aromatic plants. This steam distillation method ensures a pure and potent tulsi essential oil that retains most of the plant’s beneficial properties [10,11]. Below is a step-by-step guide for extracting tulsi oil through this method.

Steps for Steam Distillation

1.   Preparation of Plant Material

Harvesting: Choose fresh, young tulsi leaves for the best oil yield and quality. Dried leaves can also be used, though fresh leaves tend to yield more essential oil.

Cleaning: Wash the leaves thoroughly to remove any dirt or contaminants.

Cutting: Chop or bruise the leaves slightly to release their essential oils more easily during distillation. This step can help increase the surface area of the leaves, improving extraction efficiency[11].

2.   Setting up the Distillation Apparatus

Place the distillation unit (typically consisting of a boiling flask, condenser, and receiving flask) on a stable surface, preferably with heat control. Add water to the boiling flask, enough to generate steam but not so much that it will overflow. Place the chopped tulsi leaves into the distillation chamber. Ensure that the plant material is not submerged in the water, as the steam will pass through the leaves to extract the essential oil[12].

3. Initiating the Steam Distillation

Heat the water in the distillation unit. As the water heats up, it will begin to produce steam.The steam passes through the tulsi leaves, breaking the oil glands and releasing the volatile essential oils into the steam. The steam, along with the essential oils, travels through the condenser where it cools and condenses into liquid form[13].

4. Collection of Distilled Oil

The condensed mixture of water and oil flows into the receiving flask. Since essential oils are less dense than water, they will float on top of the water. The water collected in the flask is called hydrosol (or tulsi water), and the essential oil floats on top. The oil can be separated from the water using a separating funnel or by decanting the oil carefully[14].

5.   Post-Distillation Steps

After the distillation process, allow the oil to cool to room temperature. Filter the oil to remove any remaining plant debris if necessary.

Storage: Store the tulsi essential oil in an airtight, dark glass bottle to protect it from light and oxidation. Keep it in a cool, dark place to maintain its potency[14,15].

 

 

Figure 3: Steam Distillation of Tulsi leave

Characterization of Tulsi oil

1.   Qualitative phytochemical analysis

The extract was tested following standard biochemical methods as described below.

Test for proteins:

Biuret’s test: 2ml of Biuret reagent was added to 2ml of extract. The mixture was shaken well and warm for 5 min. Appearance of red or violet colour indicated presence of proteins.

Million’s test: Crude extract was mixed with 2ml of Millon’s reagent, if precipitate appeared which turned red on gentle heating confirmed the presence of protein.

Ninhydrin test: Crude extract was mixed with 2 ml of 0.2% solution of Ninhydrin and boiled for some time, if violet colour appeared indicating the presence of amino acids and proteins[17].

Test for carbohydrates:

Fehling’s test: Equal amount of Fehling A and Fehling B reagents were mixed and 2ml of it was added to the plant extract and then gently heated the sample. Appearance of brick red precipitate indicated the presence of reducing sugars.

Benedict’s test: Crude extract when mixed with 2ml of Benedict’s reagent and boiled, a reddish brown precipitate formed which indicated the presence of the carbohydrates.

Molisch’s test: 2ml of Molisch’s reagent was added to 0.5 ml of crude extract and the mixture was shaken properly. After that, 2ml of concentrated H2SO4 was poured carefully along the side of the test tube. Appearance of a violet ring at the interface indicated the presence of carbohydrate.

Iodine test: 2ml of iodine solution was mixed with 0.5 to 1 ml of crude extract. A dark blue or purple coloration indicated the presence of the carbohydrate[,18].

Test for phenol: 2 ml of alcohol and 2-3 drops of ferric chloride solution was added to 1 ml of crude extract, blue-green or black coloration indicated the presence of phenols.

Test for tannin: 1 ml of distilled water and 2-3 drops of ferric chloride solution was added to

0.5 ml of crude extract. A black coluration indicated the presence of tannin[17,18].

Test for flavonoids

Shinoda test: Crude extract was mixed with small amount of magnesium and concentrated HCl was added drop wise. Appearance of pink scarlet colour after few minutes indicated the presence of flavonoids.

Alkaline reagent test: 0.5 ml of crude extract was mixed with 2ml of 2% solution of NaOH.

An intense yellow colour was formed which turned colourless on addition of few drops of diluted acid which indicated the presence of flavonoids.

Test for saponins: 1ml of crude extract was mixed with 5ml of distilled water in a test tube and it was shaken vigorously. The formation of stable foam was taken as an indication for the presence of saponins[17].

Test for glycosides

Liebermann’s test: Crude extract was mixed with each of 2ml of chloroform and 2ml of acetic acid. The mixture was cooled in ice. Carefully concentrated H2SO4 was added. If colour change from violet to blue to green which indicated the presence of steroidal nucleus, i.e., glycone portion of glycoside.

Salkowski’s test: 2ml of chloroform was mixed with crude extract. Then 2ml of concentrated H2SO4 was added carefully and shaken gently. A reddish brown colour indicated the presence of glycoside.

Keller-kilani test: 0.5 ml of crude extract was mixed with 2ml of glacial acetic acid containing 2-3 drops of 2% solution of FeCl3. Then 2ml of concentrated H2SO4 was poured into the mixture. A brown ring at the interface indicated the presence of cardiac glycosides[18].

Test for steroid

(i)    2ml of chloroform was added to the crude extract of Tulsi. Then 2ml of each of concentrated H2SO4and acetic acid were added into the mixture. The presence of steroids was indicated by appearance of a greenish coloration in the reaction mixture.

(ii)      Crude extract was mixed with 2ml of chloroform and gently added concentrated H2SO4. A red colour was seen in the lower layer this indicated the presence of steroids.

Test for terpenoids: Crude extract was mixed in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated H2SO4 was added and heated for about 2 minutes. Presence of terpenoids was indicated by a greyish colour at the interface[17].

Test for alkaloids: 2ml of 1% HCl was mixed with crude extract and heated gently. After heating, Mayer’s And Wagner’s reagents were added to the mixture. If precipitate was observed in the reaction mixture which indicated the presence of alkaloids.

Test for anthraquinone: 5ml of chloroform and 5 ml of ammonia solution was added to 0.2 gm of plant extract. Appearance of pink, red or violet colour indicated the presence of anthraquinone.

Oils & Fats: A small quantity of crude extract was pressed between two filter papers separately. An oily appearance on filter paper indicated the presence of fixed oil and fats.

Test for lactones

Baljet’s test: Crude extract was treated with sodium picrate solution. Presence of lactone was observed by appearance of yellow to orange colour in the mixture[18].

2.   Physical Parameters Analysis

a)  Organoleptic

The physical properties of PEO were observed without changing the identity. Physical properties include smell, taste, and color. The determination of color was carried out by taking a sample of 10mL into a test tube, leaning it on a whiteboard, and observing directly[17].

b)  Determination of Relative Density

The pycnometer is filled with distilled water at a temperature of 25±0.2ºC, closed, and weighed.

The pycnometer is emptied, washed, and dried. The pycnometer was filled with 1mL of PEO at a temperature of 25±0.2ºC, closed, and weighed, calculating the specific density of PEO[16].

a)  Determination of Refractive Index

Dripped oil on the prism at a temperature of 20°C, then adjusted the slides, a clear dark and bright outline was obtained[16].

b)  Determination of Solubility in Alcohol

A total of 1mL of PEO, 70% ethanol is added drop by drop at 20°C, and each addition is shaken until the solution is as clear as possible, if the solution is not clear, compare it with the turbidity in 70% ethanol[17].

c)  Determination of Acid Value

PEO (0.5mg) dissolved in 10ml of ethanol and 2-3 dropsof PP, then titrated with a standard 0.1N potassium hydroxide solution[19].

d)Determination of Esters Value

25ml of 0.5 N KOH in alcohol, then reflux for 1h. After that, add 10ml of distilled water. Add a few drops of PP and titrate it against 0.5 N HCl[18].

e)   Determination of Optical Rotation

2 dm-long polarimeter tube was read at 20°C using D-line polarized sodium light. Different concentrations of oil solutions were prepared in ethanol[18].

f)    Determination of Iron

Determination of iron (Fe) was carried out using dry destruction analysis, by weighing a sample of 0.5grams in a porcelain dish heated at a temperature of 800°C for 2hours using a furnace and storing it in the furnace for one hour. After the cold sample was added 5ml of concentrated HNO3 and heated until dissolved and cooled. After that, 10ml of distilled water was added,bfiltered with Whatman paper, and then analyzed by an AAS (Atomic Absorption Spectrophotometer)[19].

1.   Quantitative analysis of phytochemical in the plant extract

Determination of total phenolic contents (Singleton et al., 1999)

The amount of total phenol for aqueous, methanol and ethanol extract were determined by Folin Ciocalteu reagent method. 2.5 ml of 10% Folin- Ciocalteu reagentand 2 ml of 2% Na2Co3 were added to 0.5 ml of plant extract. The mixture was then incubated at room temperature for 30 minutes. Gallic acid was used as standard (1mg/ml). The absorbance of the sample was measured at 765nm. All the tests were done in triplicates and the results were determined from standard curve and were expressed as gallic acid equivalent (mg/g of extracted compound)[20].

Determination of alkaloid (Harborne, 1973)

5 g of the sample was taken and 200 ml of 10% acetic acid in ethanol was added to the sample and allowed to stand for 4 hours. Then the solution was filtered and the extract was concentrated on water bath Conc. NH4(OH) was added drop wise and the whole solution was allowed to settle and the precipitate was then washed with dilute ammonium hydroxide and filtered. The residue was dried and weighed and this was the amount of alkaloid present in the plant material. 10 g of plant sample was taken and extracted repeatedly with 100ml 80% methanol. Then the solution was filtered and the filtrate was transferred into an empty crucible and evaporated into dryness over water bath and weighed. The final weight dry weight was amount of flavonoids in the plant sample[21].

3. Gas Chromatography analysis

Gas Chromatography (GC) is an analytical technique used to separate, identify, and quantify compounds that can be vaporized without decomposition. It is particularly useful for analyzing essential oils because these oils are composed of volatile organic compounds.In this study, GC was used to analyze the chemical composition of the essential oils extracted from Ocimum tenuiflorum[22].

Working Principle

The sample (essential oil) is vaporized and injected into the GC system. It is carried by an inert gas (usually helium) through a long, thin capillary column coated with a stationary phase. As the sample travels through the column, its components separate based on their boiling points and interactions with the stationary phase. Each compound exits (elutes) the column at a different time, called the retention time. These are detected by a Flame Ionization Detector (FID), which generates a peak for each component. The area under each peak is proportional to the concentration of the compound[23].

 

Why GC is used in this study?

GC helps identify and quantify major constituents in the essential oils, such as eugenol, thymol, linalool, etc. The retention times are compared with standards or literature values to determine which compounds are present.

Key Advantages

●  High sensitivity and precision

●  Fast and accurate identification

●  Suitable for complex mixtures like essential oils

Fresh leaves of Ocimum tenuiflorum were collected from a local botanical garden during early morning hours. The leaves were washed thoroughly with distilled water and shade-dried at room temperature (25–30°C) for 5–7 days. Once completely dried, the leaves were crushed using a mortar and pestle and stored in airtight containers until extraction[24].

 

Table 2: Condition for gas chromatography

Parameters	Value
GC system	Agilent 7890 B
Column	HP-5 (30m×0.25mm,0.25mm)
Carrier gas	Helium
Flow rate	1ml/ min
Injector temperature	250°C
Detector temperature	280°C
Injection Volume	1microliter (split 1:20)
Oven program	60°C (2min), ramp to 220°C hold 10 min.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The essential oils obtained from Ocimum tenuiflorum and were analyzed using Gas Chromatography equipped with a Flame Ionization Detector (GC-FID)[25].

Procedure

Approximately 1 µL of the essential oil sample was diluted in a suitable solvent (such as hexane) and injected into the GC system. The separation of volatile components was carried out on an HP-5 capillary column (30 m × 0.25 mm, 0.25 µm film thickness). Helium was used as the carrier gas at a constant flow rate of 1 mL/min.

The oven temperature was programmed as follows: initially held at 60°C for 2 minutes, then increased at a rate of 4°C per minute until reaching 220°C, and held for 10 minutes to ensure complete elution of all components. The injector and detector temperatures were maintained at 250°C and 280°C, respectively[].

The separated components produced individual peaks on the chromatogram, and the retention times of these peaks were compared with known standards and literature data to identify the major constituents present in the essential oils[26].

 

 

Figure 4: Schematic Diagram of a Gas Chromatography System

 

Components:

1. Carrier Gas Cylinder – Supplies inert gas (e.g., helium or nitrogen)

2.   Injection Port – Where the liquid sample is introduced and vaporized

3. Column Oven – Contains the capillary column and controls temperature programming

4. Capillary Column – Long, coiled tube coated with stationary phase to separate compounds.

5. Detector (FID) – Identifies compounds based on ionization and produces chromatographic peaks

Data System – Records and analyzes output signals as a chromatogram[27]

Results and Discussions

1.   Qualitative phytochemical analysis

Table 3: Qualitative phytochemical screening methanol extract of tulsi leaf

Phytochemicals	Results
Protein	-
Carbohydrate	-
Phenol	+
Tannin	-
Flavonoid	+
Saponin	-
Glycosides	+
Steroid	-
Terpenoid	-
Alkaloid	+
Anthraquinone	-
Fixed oils and fatty acid	-
lactones	-

 

 

2. Quantitative analysis of phytochemical in the plant extract

Table 4: Percentage of total phenolic, alkaloid and flavonoid contents in plant extract

 

 

3.         Gas Chromatography Analysis

The essential oils extracted from Ocimum tenuiflorum were analyzed using GC-FID to determine their chemical composition. The resulting chromatogram displayed multiple peaks, each representing a distinct compound separated based on retention time.

 

Table 5: Major compounds in O. tenuiflorum

Sr. No.	Name	RT (min)	Area[mV*s]	Area%
1	Eugenol	1.4333	5574.4868	83.08
2	Terpiniolene	1.5833	8.3019	0.12
3	Limocene	3.1667	13.3586	0.20
4	Sabinene	4.8000	162.3944	2.42
5	Citronellel	6.1833	411.2910	6.13
6	Isocaryophyllene	6.5500	20.8030	0.31
7	Iso-eugenol	8.5833	26.6934	0.40
8	Dimethylbenzene	10.0000	9.1565	0.14
9	Camphene	11.6500	12.6529	0.19
10	terpeneol	12.9667	11.7564	0.18
11	Nerolidol	13.6500	18.4299	0.27
12	Geraneol	13.8667	24.3816	0.36
13	Carvacrol	13.9500	38.3300	0.57
14	Humulene	14.6167	80.6844	1.20
15	α-amorphene	14.8500	42.6758	0.64
16	Luteolin	15.0000	25.3484	0.38
17	Orientin	15.3667	50.5413	0.75
18	Urosolic acid	15.5667	52.6251	0.78
19	Isorientin	15.6333	16.3781	0.24
20	Aesculin	15.8000	7.9077	0.12
21	Oglucuronide	15.8500	12.5201	0.19
22	Circimaritin	16.5333	14.9688	0.22
23	Isothymusin	16.7000	13.7894	0.21
24	Rosameric acid	16.9667	60.6966	0.90
Sum			6710.1719

 

 

Interpretation of GC Results

The GC chromatographic analysis of the Tulsi (Ocimum sanctum) extract used in the herbal antifungal patch revealed a rich phytochemical profile with 17 distinct peaks, indicating the presence of multiple volatile bioactive compounds. The most intense peak at 1.453 minutes suggests the dominance of a key constituent—likely eugenol, which is known to be the principal antifungal and antimicrobial compound in Tulsi. Other significant peaks at retention times 3.186, 4.800, 6.183, and 8.053 minutes may correspond to additional bioactive components such as methyl eugenol, caryophyllene, and ursolic acid derivatives, all of which have been reported in previous phytochemical studies of Tulsi. The distribution of these peaks reflects the complex chemical makeup of Tulsi essential oils and supports its traditional use in antifungal therapies. The presence of these compounds confirms the potential effectiveness of the prepared formulation and aligns with literature supporting Tulsi’s broad-spectrum antifungal properties.

Figure 5: Gas Chromatography of Ocimum tenuiflorum

 

CONCLUSION

This study successfully demonstrated the isolation and characterization of essential oils from Ocimum tenuiflorum and using the Steam distillation method followed by Gas Chromatographic (GC-FID) analysis.

 

SUMMARY

This research explores the isolation and extraction of essential oils from Ocimum tenuiflorum and by using steam distillation. Steam distillation is a traditional method for essential oil extraction, particularly suitable for aromatic and medicinal plants. The species of Ocimum have rich ethnopharmacological histories. Holy Basil is highly revered in Ayurvedic medicine for its adaptogenic properties and is used in the treatment of stress, inflammation, and infections. For Characterization, the essential oils obtained from Ocimum tenuiflorum were analyzed using Gas Chromatography equipped with a Flame Ionization Detector (GC-FID).

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