INTRODUCTION:

Carrot (Daucus Carota Linn., Apiaceae) is one of the most commonly used vegetables for human nutrition. It is a good source of dietary fiber and of the trace mineral molybdenum, rarely found in many vegetables. Molybdenum aids in the metabolism of fats and carbohydrates and is important for the absorption of iron. It is also a good source of magnesium and Manganese. Magnesium is needed for bone, protein, making new cells, activating B vitamins, relaxing nerves and muscles, clotting blood, and energy production; secretion and functioning of insulin also require magnesium^1-3. Carrot is native to Western or Near East Asia and is also found in the Mediterranean region, Southwest Asia, Tropical Africa, Australia, and North and South America⁴. It is considered a serious weed in Afghanistan, Greece, Hungary, and Poland, a principal weed in Jordan, Mauritius, Puerto Rico, Sweden, and Tunisia, a common weed in Austria, Canada, Egypt, England, Germany, Iran, Iraq, USA, USSR, and West Polynesia⁵. The carrot is an herbaceous, biennial plant with a height of 0.3 and 0.6 m; roughly hairy with a solid stem. In the first growing season, it shows a rosette of leaves during the spring and summer and builds up the stout taproot to store large amounts of sugars and nutrients for the production of flowers and seeds in the second year⁶. The plant produces an edible root and a leafy top within the first year. If left in the ground for another year its flower and seeds are produced⁷. Its fruits are oval and flattened from the sides, 2-4mm with short styles and hooked spines⁸. The main chemical constituents of the oil extract include β-bisabolene, camphene, β-pinene, sabinene, myrcene, γ-terpinene, limonene, α-pinene, Geranyl acetate, and carotene⁹.

Carrot seed oil is the extract of the seed from the carrot plant Daucus carota. L. The oil has a woody, earthy sweet smell¹⁰ and is yellow or amber-colored to pale orange-brown in appearance. The pharmacologically active constituents of carrot seed extract are three flavones: luteolin, luteolin 3'-O-beta-D-glucopyranoside, and luteolin 4'-O-beta-D-glucopyranoside¹¹. (Rather than the extract the distilled (ethereal) oil is used in perfumery and food aromatization. The main constituent of this oil is carotol. Pressed carrot seed oil is extracted by cold-pressing the seeds of the carrot plant. The properties of pressed carrot seed oil are quite different from those of essential oil¹². It has been used in cosmetics for its alleged anti-aging properties¹³. So keeping in view the importance of the uses of this plant an attempt has been made here to determine the characteristics of oil and standardize it for quality evaluation by using the battery of parameters.

MATERIAL AND METHODS:

The crude drug Daucus carota L. (seeds) was purchased from the local market supplier, Delhi. Carrot seeds were transported to the laboratory in glass jars and held at room temperature in the laboratory. They were cleaned with an air screen cleaner to remove all foreign matter such as dust, dirt, and broken seeds were discarded as well. Seeds were dried to a constant weight at room temperature for analysis. Seeds were crushed and made into fine powder by grinding machine.

For the oil extraction, the crushed drug material 150gm of carrot seed was placed inside a thimble made from thick filter paper, which was loaded into the main chamber of the Soxhlet extractor. 250 ml of petroleum ether was poured into a distillation flask and the Soxhlet extractor was placed onto this flask which was equipped with a condenser. The solvent was heated to reflux at a constant temperature of 68^oC and the vapor traveled up a distillation arm into the chamber housing the thimble of solid where the vapor is condensed and the warm solvent flows under gravity and percolates through the bed of the crushed carrot seed to extract the oil. When the solvent chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back to the distillation flask. The cycle was repeated for 8 hours. After the extraction, the extract was separated from the solvent via a rotary evaporator. The extract was evaporated in a vacuum. The lipid extract was collected in a flask. The extracted lipid was weighed to determine the oil content and stored under nitrogen at 4^oC for further analysis. The oil obtained from Daucus carota L. was utilized to determine its physical and chemical characteristics with the identification of fatty acids by using gas chromatography.

Estimation of essential oil:

Dried carrot seeds were ground into small pieces and subjected to hydrodistillation in the Clevenger- apparatus¹⁴ with a mixture of water and glycerine, collecting the distillate in a graduated tube in which the aqueous portion of distillate was automatically separated and returned to the distilling flask, and measuring the volume of the oil. The content of the oil is expressed as a percentage v/w¹⁵.

Physicochemical properties of seed and oil:

The weight of 1000 seeds was determined. The chemical and physical properties (moisture, crude protein, crude oil, crude fiber, total ash, acid-insoluble ash, relative density, refractive index, acid value, iodine value, free fatty acids, peroxide value, saponification number, ester value, unsaponifiable matter, etc. were analyzed as per described in the book¹⁶.

Determination of Physical constants:

Refractive Index:

The refractive index (RI) of a substance with reference to air is the ratio of the sine of the angle of incidence the sine of the angle of refraction, of a beam of light passing from air into the substance. It varies with the wavelength of the light used in its measurement. The refractive index is measured at 25° (±0.5) with reference to the wavelength of the D line of sodium.

Weight per Millilitre and Specific Gravity:

(a) Weight per milliliter:

The weight per milliliter of liquid is the weight in a gram of 1ml of a liquid when weighed in air at 25°. Determine the weight per millimeter divided by the weight in the air, expressed in g, of the quantity of liquid that fills the pycnometer at the specified temperature, by the capacity expressed in ml, of the pycnometer at the same temperature.

(b) Specific gravity:

The specific gravity of a liquid is the weight of a given volume of the liquid at 25°C. Proceed as described under wt. per ml. Obtain the specific gravity of the liquid by dividing the weight of the liquid contained in the pycnometer by the weight of water contained, both determined at 25°C.

Chemical analysis:

Determination of saponification Value:

Five (5) gram of the oil sample was weighed into a 50ml conical flask and 0.5N KOH was added to the sample, a reflux condenser was attached to the flask and heated for 30 minutes for perfect dissolution of the sample. It was allowed to cool then 1ml of phenolphthalein indicator solution was added and the content was titrated with 0.5N hydrochloric acid to an endpoint

(a) The same process was repeated using a blank sample

(b) The saponification value was calculated using the formula;

Saponification Value =

(b-a) x 0.02805 x 1000/Weight (in gram) of substance

Determination of Iodine Value:

The iodine value of a substance is the weight of iodine absorbed by 100 parts by weight of the substance. Place the substance accurately weighed, in a dry iodine flask, add 10ml of carbon tetrachloride, and dissolve. Add 20 ml of iodine monochloride solution, insert the stopper, previously moistened with a solution of potassium iodine, and allow it to stand in a dark place at a temperature of about 17° for 30 minutes. Add 15ml of the solution of potassium iodide and 100ml water; shake, and titrate with 0.1 N sodium thiosulphate, using the solution of starch as an indicator. Note the number of ml required (a). At the same time carry out the operation in the same manner, but without the substance being tested, and note the number of ml of 1N sodium thiosulphate required (b). Calculate the iodine value from the formula:

(b -a ) x 0.01269

Iodine value = --------------------------- x 100

Here W is the weight in g of the substance taken. The approximate weight, in g, of the substance to be taken may be calculated by dividing 20 by the highest expected iodine value. If more than half the available halogen is absorbed, the test must be repeated, with a smaller quantity of the substance being used.

Determination of Peroxide Value:

Place accurately weighed about 5gm oil in 250ml glass stopper conical flask, add 30ml of a mixture of 3 volumes of glacial acetic acid and 2 volume of chloroform, swirl until dissolved, and add 0.5ml of saturated potassium iodide solution. Allow to stand exactly for one minute, with occasional shaking, add 30 ml of water and titrate gradually with, with continuous and vigorous shaking, with 0.01 M sodium thiosulphate until the yellow color almost disappears, add 0.5ml starch mucilage, and continue the titration, shaking vigorously until the blue color just disappears

(a) Repeat the operation without the substance being examined

(b) Calculate the peroxide value from the following expression.

Peroxide value = (a-b) x 10 / W

Where

W = (weight in grams) of substance

Determination of unsaponifiable matter:

Introduce 5grams of the substance being examined into a 250ml flask, add 50ml of 2M ethanolic potassium hydroxide, and heat on a water bath under a reflux condenser for one hour, with frequent shaking. Wash the contents of the flask into a separating funnel with the aid of 100ml of water, and while the liquid is still slightly warm extract carefully with three quantities, each of 100ml of ether. Mix the ethereal solution in a separating funnel containing 40ml of water, swirl gently for a few minutes, and allow separating and discarding the aqueous layer. Wash the ethereal solution with two quantities, each of 40ml, of water and then with three quantities each of 40ml of a 3% w/v potassium hydroxide solution, each treatment is followed by washing with 40ml of water. Finally wash with the ethereal solution with successive quantities, each of 40 ml, of water until the aqueous layer is no longer alkaline to the phenolphthalein solution. Transfer the ethereal solution to a weighed flask, washing out the separating funnel with ether. Distil off the ether and add to the residue 6ml of acetone. Now remove the solvent completely from the flask. Dry at 100^°to 105^°C, allow to cool, place in a desiccator and reweigh. Dissolve the residue in 20ml of ethanol (96%) previously neutralized to dilute phenolphthalein solution, and titrate with 0.1M ethanolic sodium hydroxide. If the amount of 0.1M ethanolic sodium hydroxide exceeds o. 20ml, the amount weighed cannot be taken as an unsaponifiable matter, and the test must be repeated.

Determination of fatty acid:

Fatty acids were derived using the boron trifluoride method¹⁷. The fatty acids were converted to their methyl esters by heating in 10% BF₃-methanol¹⁸. Commercial mixtures of fatty acid methyl esters were used as reference data for the relative retention times¹⁹. Results are given as mean values of two replicates.

RESULTS:

Data were reported as mean ± Standard deviation. The results of various parameters are given below.

Powdered analysis:

Table No 1: Reaction of chemicals with the crude powdered drug of seeds

S. No.	With conc. Sulphuric acid	Brown
1	With acetic acid	Pale yellow
2.	With Iodine solution	Yellow
3.	With 5%ferric chloride	Green
4.	With Nitric acid	Reddish orange
5.	With 10% Sodium hydroxide	Light brown
6.	With ammonia solution	yellow
7	With Million reagent	Yellow
8.	With a 10% sodium hydroxide and a few drops of copper sulfate	Light brown
9	With conc. Sulphuric acid	Brown

Table No. 2: Fluorescence analysis of the powdered drug.

S. No	Chemical treatment	Observation
S. No	Chemical treatment	U. V. Light	Day Light
1	Drug mounted in nitrocellulose in amyl acetate	Yellowish green	Light brown
2.	Drug mounted in 0.5N Sodium hydroxide in methanol	Brownish green	Light brown
3.	Hydrochloride acid (1N) treatment	Greenish brown	Brown
4.	Drug mounted in 50% nitric acid	Dark brown	Dark brown
5.	Drug mounted in 50% Sulphuric acid	Dark brown	Greenish brown
6.	Drug as such	Light green	Light yellow

Successive Extractions of Seeds:

Successive extractive values were obtained in different solvents as per increasing orders of their polarities with the help of the Soxhlet apparatus²⁰.

Table No. 3: Extractive Values

S. No	Solvents used	Results (%) (Mean Values)
S. No	Solvents used	Sample I	Sample II	Sample III	Mean + SD
1.	Petroleum ether (60-80⁰C)	6.40	5.98	6.09	6.16+ 0.18
2.	Chloroform	2.08	2.50	3.20	2.59 + 0.51
3.	Ethyl alcohol	5.50	5.80	5.09	7.13+ 0.23
4.	Distilled water	11.80	10.98	11.50	14.10+ 0.30

Table No. 4: Physicochemical constants of carrot seeds

S. No	Parameters	Mean ± SD (%)
1	Crude protein (Nx6.25) (%)	18.50±1.20
2	Crude oil (%)	6.50 ± 0.70
3	Crude fiber (%)	10.20±2.21
4	Total ash (%)	9.52± 0.16
5	Moisture (%)	5.80 ± 0.75
6	N/10 Hydrochloric acid insoluble ash (%)	0.006 ± 0.0015
7	Weight of 1000 seeds (g)	2.59 ± 0.20
8	Essential oil yield (%)	0.83 ± 0.02
9	Alcohol soluble extractives	22.20 ± 1.2
10	Water soluble extractives	12.48 ± 2.4
11 (a) (b)	pH values 1% aqueous solution 10% aqueous solution	6.9 6.8
12	Solid contents	89.50± 2.50

Table No. 5: Elemental Analysis by AAS

S. No	Elements	Concentrations (ppm)
1	Aluminium (Al)	31.5
2	Phosphorous (P)	62.20
3	Calcium (Ca)	180.17
4	Potassium (K)	125.5
5	Sodium (Na)	32.35
6	Magnesium (Mg)	22.81
7	Iron (Fe)	20.80
8	Zinc (Zn)	0.46
9	Manganese (Mn)	0.29

Qualitative Organic constituents:

Table No. 6: Qualitative phytochemical Screening of seeds

S. No	Chemical Constituents	Test/Reagent	Result
S. No	Chemical Constituents	Test/Reagent	Pet. ether	Chloroform	Ethanol
1	Glycoside	Liebermann test	-ive	+ive	+ive
2	Sterol	Salkowaski Reaction	+ive	+ive	-ive
2	Sterol	Liebermann Burchard's test	+ive	+ive	-ive
3	Phenolics	Phenol	-ive	+ive	+ive
4	Tannins	Ferric chloride test	-ve	+ive	+ive
5	Resins	Acetic Anhydride test	+ive	+ive	+ive
6	Amino acids	Ninhydrin Solution	-ive	- ive	+ive
7	Saponins	Sodium bicarbonate solution	-ive	+ive	+ive
8	Protein	Xanthoproteic test Biuret’s test	-ive	- ive	+ive
9	Flavonoids	Magnesium Chips test	-ive	+ive	+ive
10	Triterpenes	Foam test	+ive	- ive	- ive
11	Alkaloids	Dragondroff’s reagent	-ive	+ive	- ive

Indications: (-ve) Absent and (+ve) Presence of constituent

Table no. 7: Quantitative Estimation of Phyto-Constituents

S. No	Phyto-Constituents	Mean ± SD (%)
1.	Total phenolics	1.17+ 0.07
2.	Tannins	0.17+ 0.02
3.	Resins	6.18+ 0.28
4.	Alkaloids	0.15+ 0.28

Analysis of Oil:

Organoleptic characteristic:

The preparation is oily liquid somewhat viscid in appearance, nearly pale yellow, and with persistent smell.

Physical constants:

Refractive index 1.470

Specific gravity (gram/ml) 0.9796

Titrimetric estimations:

Table No. 8: Physico-chemical properties of carrot seed oil

S. No.	Parameters	Mean Value±SD
1.	Iodine value	100.50± 2.1
2.	Acid value	4.85±1.2
3.	Peroxide value (mq/kg)	12.5± 2.1
4.	Saponification number	138.5 ± 7.5
5.	Unsaponifiable matter (g/kg)	10.2 0 ± 1.2
6.	Ester value	133.65± 9.30

Chromatographic estimation:

In the chromatographic estimation, Petroleum ether extract was used to carry out thin-layer chromatography employing different solvent systems to determine the R_f values which have been shown below.

Table No. 9 : TLC profile of different Solvent extracts of Seeds & its volatile oil

Extracts/ solvent systems

Rf values of spots observed under UV

Rf values

254nm

366nm

Spray/Treatment

Pet. ether extract

Pet. ether: Diethyl ether (9:1)

0.18 (violet)

0.30 (Pink)

0.40 (violet)

0.30 (purple) 0.40(light violet)

with 2% Ethanolic Sulphuric acid

0.40 (black)

0.69(brown)

0.85(Brownish black)

Chloroform Extract

Chloroform: Methanol (19: 1)

0.35 (violet)

Exposed to I₂ Vapours

0.30 (Yellow)

0.88 (Yellow)

0.92 (Brownish blue)

Ethanol extract

Toluene: Ethyl acetate: Formic acid (8: 1: 1)

0.15 (purple)0.30 (violet)

With 5% Vanillin Sulphuric acid(Ethanolic)

0.18 (brown)

0.36 (grayish black)

Extracted oil

Pet. ether: Diethyl ether (9:1)

0.08 (Grey)

0.39(light blue)

With 5% Vanillin Sulphuric acid (Ethanolic)

0.48 (Black)

0.57 (Bluish violet)

0.77 (Pink)

0.85 (Red)

Table No. 10: Fatty acid composition of carrot seed oil (mg/100g) employing GC technique

S. No	Fatty acids	Value in mg/100gram
1	Palmitic (16:0)	13.5
2	Stearic (18:0)	3.50
3	Oleic (18:1)	5.21
4	Linoleic (18:2)	11.5

DISCUSSION:

The powder of seed was treated with different chemicals and the screening for fluorescence characteristics with or without chemical treatment, powder as such, powder mounted in nitrocellulose in amyl acetate, 0.5N Sodium hydroxide in methanol, Hydrochloride acid (1N) treatment, 50% nitric acid, in 50% Sulphuric acid and their colors were observed in daylight i.e. visible region and ultraviolet light respectively as given in table no. 1 and 2. Extractive value in different solvents is also an important parameter to check the quality of the drug and any variation in the chemical constituents leads to the change in the resulting data. It helps in the determination of the adulteration and also an index of the purity of the drug. The mean percentage of extractive values determined in petroleum ether (40-60°C), chloroform, ethyl alcohol, and water using Soxhlet’s apparatus was found to be 6.61+0.18, 2.59+0.51, 7.13+0.23 and 14.10+0.30 respectively as given table no. 3. The physicochemical data of carrot seed is presented in Table 4. The mean percentage of alcohol and water soluble content were found to be 22.20±1.2 and 12.48±2.4 respectively. The amount of drug soluble in a given solvent is an index of its purity²⁰. The moisture content is a good parameter for detecting the quality of the drugs. Low or high moisture levels affect the quality of the drug and hence, its efficacy. The excessive moisture content becomes an ideal medium for the growth of different types of bacteria as well as fungi which subsequently spoil the drug. The mean percentage of the moisture content was found to be 5.80±0.75%. Ash values of the drug are an important parameter for the detection of impurities and adulteration. It usually represents the inorganic salts naturally occurring in the drug and adhering to it but it may also include inorganic matter added for adulteration. An ash determination furnishes a basis for judging the identity and cleanliness of the drug and gives information related to its adulteration with inorganic matter²⁰. A high ash value is indicative of contamination, substitution, adulteration, or carelessness in preparing the formulation for marketing. The mean percentage values of the total ash and acid insoluble ash were found to be 9.52±0.16 and 0.005± 0.001 respectively. The determination of crude fibbers is of considerable importance for examining certain drugs and particularly of spices that are adulterated with the waste or refused material of the same drugs and spices. The mean percentage value of crude fibers value was found to be 10.20%. The pH values of 1% and 10% aqueous solution were 6.8 and 6.9 which tells that the character of the drug is acidic. The yield of crude and volatile oil was found to be 6.50 and 0.83% respectively. Quantitative elemental estimations done by Atomic absorption spectrophotometer, physiochemical screening, and phyto-constituents of carrot seeds are presented in Tables 5, 6 and 7 respectively which show that seeds are found rich in Calcium, Phosphorous, potassium, sodium, and Magnesium, Aluminium as well as very beneficial secondary metabolites present in the drug. As far as the organoleptic characteristics of oil are concerned, it was somewhat viscid in appearance, nearly pale yellow, and with persistent smell, and values of refractive index and specific gravity were found to be 1.470 and 0.9796 gram/ml respectively. In titrimetric estimations some physicochemical properties like Iodine value, Acid value, Peroxide value, Saponification number, Unsaponifiable matter, and Ester value were 100.50±2.1, 4.85±1.2, 12.5±2.1, 138.5±7.50, and 10.20± 1.2 respectively as given in table no.8.These are an important indicator of vegetable oil quality. Peroxide value is an index used to quantify the amount of hydroxides present in fats and oils which are shown to be toxic to humans and are the primary oil oxidation products formed during the initial stages of oxidation. Besides, TLC in different extracts using different solvent systems was conducted for the separation of different compounds and Rf values of developed spots have been reported as in table no. 9. The analysis of the fatty acid composition of carrot seed oil was carried out by gas chromatography as mentioned in Table No. 10 which contains approximately oleic (5.21%), linoleic (11.50%), palmitic (13.50%) and stearic (3.50%) and others were present in minor amounts. The values reported here may differ from others, probably due to different plant seeds, soil characteristics, and environmental factors.

CONCLUSION:

The study regarding the physicochemical and phytochemical screening of carrot seed and its oil was carried out to develop the standard reference of raw materials which is widely used as a single as well as for the manufacturing of compound formulations. Besides, the thin layer Chromatographic profile of raw material extract and extracted oil was also presented which is also an important parameter to identify and assessing the presence of active and other phytoconstituents in the drug so it can be helpful in the manufacturing for the production of genuine medicines and may be utilized in detecting the adulterations as well as in identifying the authenticity of the drug in the present study.

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Received on 15.10.2023 Modified on 22.11.2023

Res. J. Pharmacognosy and Phytochem. 2024; 16(1):17-22.

DOI: 10.52711/0975-4385.2024.00004