INTRODUCTION:

Carotenoids are tetraterpenes¹. They are available in fruits, vegetables², and flowers³; also, they are present in bacteria, algae, yeasts and fungi⁴. More than 650 carotenoids have been described and isolated from natural sources⁵, however, only about 60 have pro vitamin A activity^6,7.The term carotenoids is used for several related unsaturated hydrocarbon substances having the formula C₄0Hx, which are synthesized by plants but cannot be made by animals^8,5.

Carotenoids are hydrophobic molecules^9,10 with very low aqueous solubility^10; they dissolve in some organic solvents such as acetone, alcohol, ethyl ether, tetra hydro furan, and chloroform. Carotenoids are readily soluble in petroleum ether and hexane¹¹.

The group of carotenoids can be divided (1) into the oxygen-free carotenes and (2) into the xanthophyll’s, which contain oxygen in different forms, such as one or several hydroxyl or epoxy groups^12,13. Beta carotene is the most plentiful in human foods^9,14,15.

Pumpkin (a member of the family Cucurbitaceae.¹⁶) is one of the major sources of carotenoids¹⁷.

Carotenoids play different biological roles such as increasing levels of serum insulin, reducing blood glucose levels and improvement of glucose tolerance, hypocholesterolemic¹⁸, antibacterial^18,19, anti-inflammatory and antitumor¹⁸.

Carotenoids have many other roles:

-They are responsible for the color in pumpkin²⁰;So Carotenoids is also used as a Substance to color products such as juice, cakes, desserts, butter and margarine^{5,21, 22}.

- Carotenoids have antioxidant activity^23,24 by quenching free radicals and singlet oxygen^25,26. Antioxidant functions are associated with lowering DNA damage and malignant transformations²⁷. The ability of carotenoids as antioxidants may has the main role in a reduction of disease risk such as lowering the risk of heart diseases, cardiovascular disease, and certain types of cancers, enhancing the immune system and protection from age-related macular degeneration - the leading cause of irreversible blindness among the adults^27,28,29. and they have been identified as a potential inhibitor of Alzheimer’s disease^2,5,27.

In addition to the previous functions they are the principal precursor of vitamin A⁹, which is involved in vision, cell differentiation, synthesis of glycoproteins, mucus secretion from the epithelial cells, and overall growth and development of bones³⁰. Vitamin A deficiency is a common cause of blindness and infant mortality, and a major health problem in Southeast Asia, Africa, and parts of South and Central America³¹.

Vegetables are either consumed as raw or cooked form. In the raw state, most of the nutrients are retained, whereas any degree of cooking generally results in partial loss of nutrients and considerable changes in its sensory characteristics³².Loss of carotenoids during boiling food has been reported in numerous papers^32,33. But many other studies proved increase in total carotenoids, Because Boling can lead to a dissociation of antioxidants from plant matrix components, increasing the carotenoid antioxidants³⁴.

Thus, the goal of this work was to estimate carotenoid of several samples of pumpkin, then determining the effect of boiling on the carotenoid content of them.

Figure (1): Chemical structure of beta-carotene.

MATERIAL AND METHODS:

Instrumentation:

Spectrophotometer UV-VIS (model Hitachi U-1800), Sensitive balance (Sartorius), centrifuge (model Heraeus Christ Labofuge I), micropipette, pestle and mortar, tubes, volumetric flasks.

Chemicals and Reagents:

Beta-carotene (99.3%( was purchased from Zhejiang Medicine, dichloromethane (Sigma-Aldrich), acetone (Sigma-Aldrich), butylated hydroxytoluene (BHT).

Handling of samples:

Samples were collected from pumpkin available in the local market in Syria, The samples included 20 from fresh pumpkin.

Experimental Procedures:

Preparation of Beta-carotene Standard Stock Solution:

Beta-carotene stock standard solution (100)µg/ml was prepared by dissolving (1)mg of (beta-carotene) into (3) milliliters of dichloromethane in a volumetric flask (10) m. Then the solution was diluted to the volume using acetone.

Preparation of Beta-carotene Working Standard Solution: Working standard solution (30) µg/ml was prepared by taking (3) mL of the stock standard solution and diluting it to (10) mL with acetone/ dichloromethane (70/30) in a volumetric flask.

Preparation of extraction solution (dilute solution): In (500) mL volumetric flask (150) ml dichloromethane was put then the volume was made up to the mark using acetone. Add 0.05mg of BHT (butylated hydroxytoluene) as an anti-oxidant substance.

The proportion of acetone/ dichloromethane mixture was 70/30.

Procedures of boiling pumpkin:

The samples of pumpkin were put into a beaker (500 mL) and cooked with boiling tap water (200mL) for (5, 10, 15, 30, 45, 60) min at 100^◦C. Each sample was divided into six portions. Whereas the effect of time boiling on carotenoid content of pumpkin was studied. The samples included 10 from fresh pumpkin.

Extraction methods:

Due to their complex structure of carotenoids and because of the wide variety of these compounds present in vegetables and fruits, there is not a reference method to analyze them³⁵.

According to International Organization for Standardization (ISO): two routine methods are mentioned: the first one is method A, which estimate carotenoids in products having fat content of less than or equal to 5% (w/w). The determination carotenoids content depends on extracting them using organic solvent then quantify them by measuring their absorbance by visible- spectrophotometer at wavelength of 450nm.

Method B for products having a fat content of more than 5% (m/m), the saponification is necessary, which allows removing the ballast substances, especially chlorophyll, then extracting carotenoids is achieved using organic solvent and determined spectrophotometrically^1,21,35,36.

The extraction was practically achieved by using a mixture of dichloromethane/acetone in a ratio of (30/70%).

100mg of the pumpkin was placed in a mortar and crushed with a pestle. 5 ml of extracting solvents (mixture of dichloromethane and acetone in the ratio of 30:70) was added to the mortar and the sample was crushed. Then transferred into tubes and closed them well (tubes covered with a layer of aluminum foil). Tubes were centrifuged for 10 minutes at 3500rpm. Then the supernatant (completely clear) was transferred into a volumetric flask (10) mL, and diluted with extracting solvents to the volume.

After extraction, the total carotenoids content was determined immediately (because acetones is volatile) by spectrophotometer at 454 nm.

Determination the wavelength: (λ Max):

The absorbance spectrum of the solution of beta-carotene was scanned between (400-800) nm against the corresponding blank. The maximum absorbance was at 454nm^{37, 38}. The spectrum was shown in figure (1).

Fig.1: Determination of λ Max in colorimetric method.

RESULTS AND DISCUSSION:

Validation of the spectrophotometric method³⁹:

Linearity:

A six standard series was established 0.3, 0.6, 1.5, 3, 6, 9 µg/ml for beta-carotene .The absorbance at 454 nm was measured. The absorbance is plotted against the beta-carotene concentration [Figure (2)].

Fig.2: Calibration Curve of colorimetric method.

The correlation coefficient R² was 0.9986 and the equation of straight line was y =0.1763x+0.0243. Thus, the method has a good linearity.

Accuracy:

Accuracy should be reported as percent recovery by measuring three known concentrations of beta-carotene (1.2, 1.5, and 1.8) µg/ml. The percent recovery was ranged from 98.5to102.2 %.

Precision:

The relative standard deviation RSD% (coefficient of variation) was determined by measuring six solutions at the same concentration (1.5µg/ml). The relative standard deviation was 1.60%.

Sensitivity:

The detection limit (LOD) and the quantitation limit (LOQ) of beta - carotene were determined as 0.1 µg/ml and 0.3 µg/ml respectively.

Determination of carotenoid content of samples before boiling:

The samples were extracted as above mentioned and their beta-carotene contents were determined by spectrophotometric method.

The results of determination of carotenoids contents of pumpkin samples are shown in Table (1).

The carotenoids contents ranged from 80.56 to 216.46 µg/g. These values are similar to those found by Soza et al in pumpkin which ranged from 11.8 to 290.62 µg/g⁴⁰ .

The variability of the results is high, the composition of carotenoids may also be affected by some factors such as variety, cultivar, maturation stage, geography, climate, the type of sample and the part of plant, harvesting and post-harvesting^32,41.

Table (2):Percentage of decreased carotenoid content during boiling of pumpkin.

Boiling time (min.)	Sample1	Sample2	Sample3	Sample4	Sample5	Sample6	Sample7	Sample8	Sample9	Sample10
5	1.35	13.26	0.45	10.54	0.94	3.69	2.66	6.25	3.16	2.94
10	12.68	18.82	0.18	20.98	23.07	3.69	15.28	19.22	14.01	7.92
15	22.27	19.8	24.07	22.37	31.41	17.3	19.97	25.05	20.81	40.54
30	35.63	47.67	33.79	22.65	38.55	31.14	35.05	40.37	41.11	51.73
45	37.15	55.82	50.99	24.43	14.91	31.81	55.31	46.55	43.23	55.28
60	44.76	72.56	55.55	26.16	46.68	33.93	66.45	46.55	52.73	64.49

CONCLUSION:

As shown in the study above, boiling affects strongly on carotenoid contents in pumpkin, due to causing definite damage to carotenoids, which results in significant loss carotenoid contents.

CONFLICT OF INTEREST:

The authors declare that there is not any conflict of interest related to this work.

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Received on 04.11.2020 Modified on 27.12.2020

Research J. Pharm. and Tech 2021; 14(11):5642-5646.

DOI: 10.52711/0974-360X.2021.00981

Carotenoid content found µg/g	Sample cod
204.66	L1
142.32	L2
117.84	L3
110.16	L4
112.80	L5
216.46	6L
122.9	7L
105.8	L8
120.07	9L
117.8	L10
99.63	L11
80.56	L12
84.91	L13
123.4	L14
84.583	15L
84.19	L16
82.93	17L
82.34	L18
107.71	19L
110.16	L20