Chemical Composition and Antimicrobial Activity of different Solvent Extracts of Carthamus tinctorius Flowers

 

Sura L. Alkhafaji*¹, Abbass M. Kashamar², Ibrahim H. Alkhafaji¹

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kerbala University, Karbala - 56001, Iraq.

²Department of Agricultural Crops, Faculty of Agriculture, Kerbala University, Karbala- 56001, Iraq.

 

ABSTRACT:

Carthamus tinctorius is an annual plant belonging to the Asteraceae family. It is known as safflower. It is cultivated in Southern Asia, North and South America. It contains many bioactive compounds for which it has the biological activities, involving antidiabetic, anticancer, antioxidant, anti-ageing, anticoagulant, hepatoprotective and anti-inflammatory, analgesic and antibacterial activities. The goal of this work is to identify the chemical constituents of safflower petals and to reveal their antimicrobial activities. The dried petals were extracted using three solvents, hexane, ethyl acetate and methanol. Safflower was studied by gas chromatography-mass spectrometry (GC-MS). A total of 78 compounds from the three extracts were defined, and it was indicated that the highest percentage in ethyl acetate extract go to 4’, 6- Dimethoxyisoflavonne-7-O-β- D-glucopyranoside (15.6%) and 7, 4’ – dimethoxy-3-hydroxy flavone (8.17%). In methanol extract, the high percentage belongs to Ascorbic acid, per methyl- (25.16%), Papaverine (8.16%), Tetratricontane (5.99%). In contrast, Morin (14.06%) and Isolongiflol (22.67%) showed the highest concentration in hexane extract. The antibacterial activities of Carthamus tinctorius petals were evaluated against Staphylococcus aureus, Staphylococcus heamolyticus, Escherichia coli, and Pseudomonas aeruginosa by the disc diffusion method and the minimum inhibitory concentration (MIC). The results show that C. tinctorius has antimicrobial activity against all the tested bacteria with diverse degrees. The highest antibacterial activity was achieved by ethyl acetate extract against all gram-positive and gram-negative bacteria involved.

 

 

 

INTRODUCTION:

Safflower (Carthamus tinctorius) is an annual, thistle, semi-spinal plant that belongs to the Asteraceae family and it grows in a warm climate. Its flowers are yellow, orange or red (Figure 1). Initially, it is found in Southern Asia, (especially in China, India, Iran and Egypt), southern Europe, North and South America¹. The other producing regions are Turkey, Argentina, Australia, Iraq, and Russia.

 

Figure 1: The Flowers of Safflower

 

The petals, leaves and seeds oil of safflower have many applications in cosmetic, textile industry, currently as a food colorant² and in herbal and traditional medicine. Safflower was indicated to treat several diseases such as dysmenorrhea, amenorrhea³, cardiovascular^4,5,6,7, cerebrovascular⁸, the pain of joint and abdominal pain. Safflower seeds and extracts have been used to stimulate bone formation and preclude the osteoporosis occurrence. Seeds possess major antinflammotry⁹ and antioxidant^10,11,12,13, analgesic¹⁴, and antimicrobial proerties¹⁵.

 

Safflower bioactivities are attributed to the presence of chemical compounds like flavonoids, phenylethanoid, glycosides, coumarins, steroids, fatty acids and polysaccharides¹⁶

 

Seeds oils contain a considerable amount of polyunsaturated fatty acids (i.e., 70% linoleic acids), 10% oleic acid (mainly, monosaturated fatty acid) and straightforward amount of stearic acid, palmitic acid, margaric acid, etc.^17,18. Also, the seeds are rich in vitamins (Cu, Zn, Mg and Fe), minerals (B carotene, Thiamine), alpha, beta, gamma types of tocopherols. The petals produce red/yellow pigment which consists of saffiomin A and safflor yellow B)^2,19

 

According to medicinal effects and pharmacotherapeutic uses of C. tinctorius, further chemical determination is required to examine the chemical components of this plant, for that reason, this present work was aimed to extract the safflower petals of C. tinctorius using three different solvents (ethyl acetate, methanol and hexane), then the chemical constituents were identified by GC-MS technique. GC–MS is one of the most well-known analytical techniques in many scientific fields due to its high sensitivity, low detection limit, rapid identification and its ability to analyse a number of ingredients in analytes concurrently. Also, it is aimed to examine the in vitro antibacterial activity of the active agents against Staphylococcus aureus, Staphylococcus heamolyticus, Escherichia coli and Pseudomonas aeruginosa.

 

The antibacterial activity of safflower extracts against tested bacteria is attributed to phenolic derivatives that are available in the form of ester or rarely in glycosides form, flavonoids (in the form of glycosides), alkaloids, terpenoids and other bioactive compounds.

 

MATERIAL AND METHODS:

Plant sample preparation:

Safflower applied in this work was acquired from the local market. The orange flowers were washed, then they were dried in the Pharmacognosy Department, College of Pharmacy, Kerbala University, Iraq.

 

Solvents and Chemicals:

Ethyl acetate EA, methanol ME, and hexane HE were used as the extraction solvents in this study. These three analytical grade solvents were purchased from Sigma Aldrich.

 

Extraction Methods:

Three hundred grams of the dried flowers were pulverized in a mechanical grinder then extracted by the graduated method as the following:

 

A hundred gram of the dried powder of safflower petals were soaked in 600ml of n –HE for six days then the extracted product was filtered through a filter paper (Whatman no. 41), the filtrate was evaporated under a reduced pressure using a rotatory evaporator at a temperature of 40˚C to get the HE crude extract.

 

The flower residues were re-extracted by 600ml of EA solvent and macerated for another six days then filtered. The clear filtrate was again dried under a reduced pressure by rotatory evaporator at a temperature of 40˚C to yield the extract of EA.

 

The flower remains from the second extraction were re-extracted using ME solvent 600 ml. After maceration for six days, it was filtered. The filtrate was dried using a rotatory evaporator at a temperature of 40˚C to get ME extract. All the extracts were stored for GC-MS analysis.

 

GC–MS analysis for identification of C. tinctorius chemical composition:

The three extracts were examined by a GC (Agilent Technologies 7890A) interfaced with a mass- selective detector (MSD, Agilent 7000) furnished with a polar Agilent HP-5ms (5% phenyl methyl polysiloxane) capillary column (30x0.25mm i. d. and 0.25μm film thickness). The carrier gas was Helium with a linear flow of 1ml/min, and the volume of injection was 1µl of the sample. The injector temperature was 200˚C. The ionisation potential of 70 eV, interface temperature of 250˚C, and acquisition mass range 50-800 were the MS operating parameters.

 

The identification of compounds was based on comparison of their mass spectra and retention indices (RIs) with those documented in the Wiley and NIST mass spectral databases, and authentic samples (compound available in our laboratories), additional library data of the GC– MS system or from literature data.

 

Determination of antibacterial activity:

Tested Bacterial strains and culture media preparation:

The antibacterial activity of the EA, ME and HE extract of safflower was examined against two gram-positive bacterial strains (Staphylococcus aureus and Staphylococcus and two gram-negative bacterial strains which are Escherichia coli and Pseudomonas aeruginosa. These bacterial strains were isolated and acquired from the medical laboratory unit, Al-Kafeel Hospital, Karbala. All Bactria were cultured in sterilised nutrient broth (NB) at 37°C for 16-18 h. Nutrient broth (NB, 25g/L), Muller-Hinton (MH, 38g/L), was dissolved in distilled water. The tools that have been used; the glasses (pipettes, tubes, Z- rode and beakers), filter paper disc (5 mm in diameter) and the solution of NB and MH were sterilised using autoclave at 15Ibs at 121˚C for fifteen minutes. The concentration of tested bacteria, cultures were prepared by comparing with 5% McFarnald solution (0.05ml BaCl2 solution in broth, and 9.95ml of H2SO4 solution in 1% in broth) that adjusted to 150x10⁶ colony-forming unit CFU/ml. 1800µg/mL of each crude extract was prepared by dissolving 1.8mg in 1.0ml of Dimethyl sulfoxide (DMSO).

 

Antibacterial assay:

Disc Diffusion Method:

The antibacterial activity was determined by disc diffusion method and minimum inhibitory concentration (MIC) as mentioned in literature²⁰ With minor modification. The Petri dishes used (90×15mm) were spread with sterilized MH (20ml) solutions, after that 200μl of bacteria stock (150×10⁶ CFU/ml); each was spread on the Muller- Hinton agar (MH) medium using Z-glass rod, after that the two paper discs individually saturated with 20μl of extract (1800μg/mL) were applied over each of the culture plates previously planted with 150×10⁶ CFU/ml cultures of bacteria. Two blank discs (with DMSO only), standard disc of Gentamycin (10μg/disc) that was used as control for comparison, was placed and arranged on MH petri dish. All bacterial cultures were incubated at 37˚C for 24 h. After incubation the zone of inhibition around each paper disc was measured, and the antibacterial action was verified. The inhibition zone is outlined as the area with no growth of the tested infectious agent.

 

Determination of Minimum Inhibitory Concentration (MIC):

The minimum inhibitory concentration MIC of each extract was determined for each tested bacteria in duplicates. The concentration of stock solution (1800μg/mL) that prepared by dissolving of 1.8Zmg in 1.0ml of DMSO, was diluted twofold to get a concentration range of (14.07-1800μg/mL) for each sample, and then 100μl of microorganism was added to 96- wells and were enclosed for incubation overnight at 37˚C.

 

RESULTS AND DISCUSSION:

For identifying the components of safflower petals, GC-MS analysis was accomplished, and the peaks, numbers and types of its compounds were determined. The results were recorded in Table 1, 2, and 3 show that Safflower constituents of petals are very diverse, complex and have a significant difference in percentage. Hence, EA, ME and HE extract consist of 40, 35 and 27 identified compounds, respectively. These compounds are ranging from flavonoids and its glucopyranoside, terpenes, coumarins, esters, alkanes, phenols, essential oils, alkaloids, vitamins and others.

 

The major components were 4’, 6- Dimethoxyisoflavonne-7-O-β- D-glucopyranoside, 7, 4’ –dimethoxy-3-hydroxy flavone, Ascaridole, Hexa-hydro- farnesol, 9- cis Retinoic acid, 4-hydroxy-7-methoxy-3-(4-methoxyphenyl) coumarin, Gossypetin, Citronellyl tiglate, Papaverine, Tetratricontane, Gardenin, Octacosane, Phytanic acid, Morin and Isolongiflol.

 

For EA extract, the GC-MS analysis revealed 40 compounds. The dominant compounds were 4’, 6- Dimethoxyisoflavonne-7-O-β- D-glucopyranoside (15.6%) and 7, 4’ – dimethoxy-3-hydroxy flavone (8.17%). Other minor compounds were 6,4’-dimethoxy-7-hydroxyisoflavone (5.92%), isolongifolol (3.66%), ledol (3.55%), phytol (1.03%), 2’-hydroxy-2,3,4’,6’-tetra methoxy chalcone (3.01%), syringic acid (2.82%) and Ascaridole (2.82%), as listed in Table 1.

 

Table 1: Compositions of Carthamus tinctorius petals for EA extractor solvent

No	RT/min	Name	Area%
1	7.81	2'-Hydroxy-2,3,4',6'-tetramethoxychalcone	3.01
2	9.125	Syringic acid	2.82
3	9.769	Phytol	1.03
4	9.864	Himbaccol	2.34
5	10.345	2'-Hydroxy-2,4,4',5,6'-pentamethoxychalcone	1.69
6	10.489	Longiborneol	1.41
7	10.823	7,8-Dihydro-α-ionone	2.49
8	10.935	Farnesol	1.74
9	11.287	Caryophyllene	1.43
10	11.503	Ascaridole	2.82
11	11.624	Squalene	1.53
12	11.831	Dihydrophytol	1.55
13	12.066	(S)-(-)-Citronellic acid	1.51
14	12.345	Hexa-hydro-farnesol	2.82
15	12.579	Isophytol	0.81
16	12.854	Phytol	3.57
17	13.016	9-cis-Retinoic acid	2.75
18	13.137	p-Menth-8-en-2-one	2.73
19	13.268	Nerolidol	1.35
20	13.579	Isolongifolol	1.64
21	13.732	4',6-Dimethoxyisoflavone-7-O-β-D-glucopyranoside	15.6
22	13.858	cis-Z-α-Bisabolene epoxide	1.01
23	13.934	Ethyl linalool	1.83
24	14.308	Apigenin 8-C-glucoside	1.89
25	14.425	Sesquicineole	1.55
26	14.547	β-Santalol	1.18
27	14.624	trans-Geranylgeraniol	0.87
28	14.745	Isolongifolol	3.66
29	14.777	Ledol	3.55
30	14.943	6,4'-Dimethoxy-7-hydroxyisoflavone	5.92
31	15.182	α-Cadinol	0.98
32	15.596	3-Hydroxy-6,2',4'-trimethoxyflavone	0.86
33	15.848	1,8-Diazabicyclo [5.4.0] undec-7-ene	1.3
34	16.227	1-Hexacosanol	2.62
35	16.447	7,4'-Dimethoxy-3-hydroxyflavone	8.17
36	18.595	Quercetin 3,5,7,3',4'-pentamethyl ether	2.35
37	19.19	Elemol	1.24
38	21.554	4-Hydroxy-7-methoxy-3-(4-methoxyphenyl) coumarin	1.64
39	22.635	7,3',4',5'-Tetramethoxyflavanone	1.64
40	22.928	Cedrol	1.13

 

While in ME extract, the predominant compounds were aliphatic acids (31.21%) and alkanes (20.69%). The high percentage belongs to Ascorbic acid, per methyl- (25.16%), Octacosane (10.24), Papaverine (8.16%), and Tetratricontane (5.99%).and 1- naphthalenol (3.74%), caryophyllene oxide (0.62%) as listed in Table 2.

 

Table 2: Compositions of Carthamus tinctorius petals for ME solvent extractor

No	RT/min	Name	Area%
1	3.194	β-Citronellol	0.7
2	3.811	1H-Pyrazole, 1,5-dimethyl-	0.35
3	5.428	Resorcinol	0.93
4	6.252	6-Methoxyluteolin	0.7
5	6.855	Thiazolidine-2-carboxylic acid	1.62
6	7.342	1-Naphthalenol	3.74
7	7.859	2'-Methoxy-α-naphthoflavone	0.74
8	8.4	3,2'-Dimethoxyflavone	0.97
9	8.467	5,7,3',4',5'-Pentahydroxyflavone	0.88
10	12.318	Gossypetin	2.33
11	12.484	Ascaridole	0.49
12	12.588	Caryophyllene oxide	0.62
13	12.876	Citronellyl tiglate	1.11
14	13.011	5β,7βH,10α-Eudesm-11-en-1α-ol	0.41
15	13.155	Pinane	0.77
16	13.38	5,7-Dimethoxyflavone	0.5
17	13.565	Isopulegol	0.84
18	13.678	7,4'-Dimethoxy-3-hydroxyflavone	2.77
19	14.443	9-cis-Retinoic acid	0.8
20	14.731	Phytol	1.95
21	14.758	Isolongifolol	2.65
22	14.911	Papaverine	8.16
23	15.105	Gardenin	3.08
24	15.92	Tetratriacontane	5.99
25	16.393	3-Hydroxy-3',4',5'-trimethoxyflavone	0.61
26	17.348	3-(3,4-Dimethoxyphenyl)-7-methyl-4-phenylcoumarin	0.47
27	19.865	Vitamin E	0.82
28	20.532	Ascorbic acid, per methyl-	25.16
29	20.649	Squalane	3.69
30	20.73	Octacosane	10.24
31	21.383	Vitexin	1.01
32	22.081	N-Ethyl-4-piperidine carboxamide	9.01
33	22.473	Longiborneol	0.5
34	22.775	Phytanic acid	3.63
35	22.905	Geranyl isovalerate	1.75

 

As shown in Table 3, Morin (14.06%) and Isolongiflol (22.67%) compounds were the dominant compounds in HE extracts. Other important compounds were identified such as 4', 6-Dimethoxyisoflavone-7-O-β-D-glucopyranoside (3.07%), 6,7-Dihydroxy-4-phenylcoumarin (2.84%), 6,7-Diallyloxy-4-methylcoumarin (1.4%), 5,7,3',4',5'-Pentahydroxyflavone (0.56%), 7-Methoxy-4-methylcoumarin (0.34%), 3'-Benzyloxy-5,6,7,4'-tetramethoxyflavone (0.34%).

 

Table 3: Compositions of Carthamus tinctorius petals for HE extractor solvent

No	RT/min	Name	Area%
1	4.018	2-Methylthiolane	0.3
2	5.783	Methimazole	0.31
3	7.999	Resorcinol	0.26
4	8.4	7-Methoxy-4-methylcoumarin	0.34
5	9.728	3,4,5-Trimethoxycinnamic acid	0.35
6	10.07	Cyclizine	0.29
7	10.273	Caryophyllene	0.23
8	10.345	p-Cresol, 2,6-di-tert-butyl-	0.28
9	10.466	Resorcinol, 5-pentyl-	0.29
10	10.606	Camphor	0.26
11	10.759	Phenol, 2,3,5,6-tetramethyl-	0.29
12	11.07	6,7-Dihydroxy-4-phenylcoumarin	2.84
13	11.444	3'-Benzyloxy-5,6,7,4'-tetramethoxyflavone	0.34
14	11.903	L-Pinocarveol	0.31
15	12.52	Citronellyl tiglate	3.65
16	12.641	6,7-Diallyloxy-4-methylcoumarin	1.4
17	12.903	Caffeine	2.09
18	13.065	Isomenthol	1.16
19	13.186	Pinane	0.88
20	13.384	5,7,3',4',5'-Pentahydroxyflavone	0.56
21	13.515	2'-Hydroxy-2,4',5-trimethoxychalcone	0.32
22	13.862	4',6-Dimethoxyisoflavone-7-O-β-D-glucopyranoside	3.07
23	14.231	Morin	14.06
24	14.506	Thunbergol	0.86
25	14.925	Patchoulane	2.61
26	15.438	Isolongifolol	22.67
27	15.564	Genkwanin	1.46

 

This difference in the percentage and number of components that were extracted by the three extract solvents can be attributed to the polarity difference of solvent extractors.

 

Although the results showed different components and which were extracted by different solvents, some of the compounds precisely, Isolongiflol; was common in three extracts with different percentage. Meanwhile, it was detected some of compounds presented in two samples and not in others. Hence, 7, 4’ – dimethoxy-3-hydroxy flavone, Phytol, Squalane, ascaridole, 9- cis- retinoic acid were found in both EA, and ME extracts. While, 4’, 6- Dimethoxyisoflavonne-7-O-β- D-glucopyranoside, caryophyllene were common in ET and HE extracts. 5, 7,3’,4’,5’-pentahydroxyflavone, and Resorcinol compounds were founded in both HE, and ME extracts.

The antibacterial activity of the three extracts of dried petals of C. tinctorius against selected gram-positive and gram-negative bacteria was determined by a disc diffusion method and minimum inhibitory concentration (MIC). A significant effect of solvent polarity was predicted. So, as shown in Table 4, and at 1800 mg/ml, EA and ME extracts of safflower had an inhibition on all test organisms. EA manifested the main inhibitory results and ME extracts. EA solvent extract inhibited in some extent S. haemolyticus and P. aeruginosa, and ME extract on P. aeruginosa and S. aureus for which the inhibition zone (DIZ) were 12 mm and 8 mm, and 7, 5 mm, respectively. While the inhibition activity of HE extracts was the lowest against all type of selected bacteria.

 

Table 4: Antibacterial activity of different extracts of C. tinctorius petals by disc diffusion method

Tested organism	Type of Extract	Inhibition zone (DIZ) mm
Escherichia coli	EA ME HE	5 5 5
Pseudomonas aeruginosa	EA ME HE	8 7 5
Staphylococcus aureus	EA ME HE	5 5 5
Staphylococcus heamolyticus	EA ME HE	12 7 5

 

Among gram-positive bacteria, maximum antibacterial action was obtained against S. haemolyticus followed by S. aureus. While, amongst gram-negative strains, maximum activity was realized against P. aeruginosa followed by E. coli. Contrarily, HE extract showed low activity against both gram-positive and gram-negative organism because it could be inclined by the fact that the inhibition zone depends on how the antimicrobial compound can diffuse equally through the bacterial wall. Therefore, for obtaining quantitative results, it was decided to apply MIC procedure to determine the antibacterial activity.

 

The minimum inhibitory concentration (MIC) test was carried out against S. aureus, S. heamolyticus, E. coli and P.aeruginosa. MIC results were reported in Table 5. The results revealed MIC values ranging from 28.12- 900 µg/ml. The selected extracts showed the different level of the antibacterial effect depends on the tested bacterial strains. Knowing that very low concentration of extract is required to suppress the growth of bacteria.

 

According to MIC data, the results indicated that EA extract had the same bioactivity against all types of pathogens in a concentration of 28.12µg/ml. Also, ME extract inhibited S. heamolyticus, E. coli and P. aeruginosa in minimum concentration of about 112.5 µg/ml except for S. aureus; it was observed that MIC has increased up to 225µg/ml. However, non-polar solvent HE extracts had a MIC value range between 225 and 900 µg/ml against all bacterial species.

 

Table 5: MIC of EA, ME and HE extracts of C. tinctorius petals

Tested organism	Extract	MIC value Mg/ml
Escherichia coli	EA ME HE	28.12 112.5 900
Pseudomonas aeruginosa	EA ME HE	28.12 112.5 450
Staphylococcus aureus	EA ME HE	28.12 225 225
Staphylococcus heamolyticus	EA ME HE	28.12 112.5 900

 

Regarding MIC data, the bacterial inhibition activity of the three extracts against all tested organisms was in decreasing order as following: EA extract > ME extract > HE extracts. The highest antibacterial activity was achieved by EA extract against all gram-positive and gram-negative bacteria. Overall, the high MIC values of the tested bacteria in this study suggest an organism resistance to antibiotics or the plant extract is less active against pathogens, while the low MIC values for bacteria indicate the efficiency of plant extract.

 

CONCLUSION:

The findings of this study revealed that the safflower plant consists of several chemical compounds. Seventy-eight chemicals in petals part of C. tinctorius could be recognised by GC-MS analysis. The chemicals of EA, ME and HE extracts from safflower have moderate to worthy antibacterial activity against tested pathogens. Therefore, it is suggested that these extracts may help to open the door to discover new antibiotic agents to treat many infectious diseases. Further research is needed to isolate and identify other secondary metabolites from the extracts employed for testing the antibacterial activity and understanding the principal mechanisms.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Kerbala College of Pharmacy for providing all the necessary facilities to complete this work. the authors are thankful to medical laboratory unit, Al-Kafeel Hospital, Karbala for providing the bacterial strains.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 24.11.2019           Modified on 07.02.2020

Accepted on 28.03.2020         © RJPT All right reserved