Cytotoxicity Studies of the Chemical Constituents from Marine Algae Chara baltica

*Corresponding Author:

V. B. Tatipamula
K L College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram-522 502, India
E-mail: vinaybharadwajt@gmail.com

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

Abstract

For the first time, 89 chemical constituents were identified by gas chromatography-mass spectrometry analysis of ethanol extract of Clathria baltica. The chemical examination of ethanol extract of Clathria baltica yielded three known metabolites, 1, 2 and 3, for first time from the marine algae Clathria baltica. All three metabolites and the ethanol extract of Clathria baltica were screened against cancer cell lines, MCF-7, DLD-1, HeLa, FADU, A549, and SKOV3, and one normal human cell line using sulforhodamine B assay and doxorubicin as the standard. Among all isolates, compound 3 showed significant growth inhibition of MCF-7, DLD-1 and FADU with IC50 values of 26.5, 15.5 and 16.5 µg/ml, respectively. Whereas, all the metabolites and the extract exerted lower growth inhibitory effect on the normal human cell line tested. This is the first in vitro gas chromatography-mass spectrometry analysis, as well as, cytotoxicity report on marine algae Clathria baltica.

Keywords

GC-MS analysis, isolation, characterization, anticancer activity, sulforhodamine B assay

Globally, cancer still remains an aggressive killer that severely effecting the human population[1]. According to the report of the International Agency for Research on Cancer (IARC), it was documented that within 5 y of diagnosis - 9.6 million cancer deaths from 33.7 million people living with cancer has taken place and 18.1 million new cancer cases were reported in 2018 across the world[2]. In addition, the IARC is also estimated that about 26 million new cancer cases and 17 million cancer deaths per year may be recorded by 2030[3]. In addition, the present therapies for cancer include chemotherapy and radiotherapy, which cause lot of strain to patients and ultimately damage their health[4]. Hence, researchers are focussing on developing new, safer and effective drugs to treat cancer.

From the dawn of ancient medicine, natural products have been used to treat many deadly diseases like cancer. In addition, from the past 30 y, natural products have received great attention in attaining potential as well as novel cancer therapeutic agents[5,6]. Moreover, the mechanism of action of inhibiting various stages of tumorigenesis by most of the natural products were also well-documented[6].

In folklore, marine algae (seaweeds) are used as food, feed, fuel and livelihood in Asian countries. Based on the pigmentation, marine algae are classified as red, brown and green algae. From the past few decades, marine algae have been highly acknowledged to possess noticeable pharmacological activities, which include antineoplastic, antimicrobial and antiviral activities due to their specific functional compounds (which are not available in other plants)[7-10]. Particularly, marine algae of Chara genus (family: Characeae- charophyte green algae) reported to possess and antioxidant enzyme, allelophatic activities and lipid peroxidation capabilities along with high levels of photosynthetic pigment content[11]. Earlier reports from our laboratory showed phytochemical analysis along with antioxidant activities of marine algae Chara baltica[12]. Till date, complete chemical and pharmacological evaluation has not been reported on C. baltica. Based on the aforementioned properties of Chara genus, as well as, C. baltica, C. baltica specimens (in July 2018) were subjected to gas chromatography-mass spectrometry (GC-MS) analysis, chemical and cytotoxicity evaluation, which are reported in this research communication.

Materials and Methods

The specimens of seaweed Clathira baltica was collected near Korangi coast, Kakinada, Andhra Pradesh, India at a depth of 8-10 ft (16°80’N and 82°08’E with 3 m elevation) on 3 July, 2018. The seaweed was authenticated in the Botany Department, Andhra University, Visakhapatnam, India. A voucher specimen was deposited in the Marine Organisms Museum in the University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India (No. AU-SW-2018-947). All chemicals used in the present study were of analytical grade. Streptozotocin was from Himedia Laboratories Pvt. Ltd. (Mumbai, India); glibenclamide from the Aventis Pharma Ltd. (Mumbai, India); and rat feed from the Hindustan Lever Ltd. (Mumbai, India).

Extraction and isolation:

The seaweed was cleansed from extraneous substance and stored in ethanol-water (9:1) at the site of collection. The crude plant material (100 g) was extracted at least thrice with ethanol-water and concentrated at a reduced pressure and the combined extracts were lyophilized to obtain ethanol extract of C. baltica; as a reddish brown gummy residue (3.25 g). About 2 g of ethanol extract was subjected to column chromatography, afforded three fractions. Fraction I (510 mg) and fraction II (800 mg) were subjected to repeated column chromatography using hexane and ethyl acetate as a solvent system yielded 1 (240 mg) as a colourless liquid and 2 (330 mg) as a pale greenish liquid, respectively. Fraction III (400 mg) were purified by recrystallization technique using hexane and acetone as solvents yielded 3 (200 mg) as a greenish crystals.

Compound 1 (dihydrofuran-2,5-dione), colorless crystals, molecular formula: C₄H₄O₃; yield: 240 mg; R_f: 0.7 (hexane:ethyl acetate, 1:9); ¹H NMR (CDCl₃, 400 MHz) δ 4.00 (4H, s, 2,3-CH₂); ¹³C NMR (CDCl₃, 400 MHz) δ 22.16 (C-2/C-3), 170.24 (C-1/C-4); LCMS m/z: 100.

Compound 2 (3-hydroxy-4,4-dimethyldihydrofuran- 2(3H)-one), pale greenish liquid, C₆H₁₀O₃; yield: 330 mg; R_f: 0.6 (hexane:ethyl acetate, 1:4); ¹H NMR (CDCl₃, 400 MHz) δ 1.00 (6H, s, 5,6-CH₃), 2.31 (1H, s, 2-CH), 3.86-3.88 (1H, d, J= 8 Hz, 4a-CH₂), 4.04- 4.06 (1H, d, J= 8 Hz, 4a-CH₂); 4.11 (1H, s, 2-OH); ¹³C NMR (CDCl₃, 400 MHz) δ 23.15 (C-5/C-6), 43.40 (C-3), 80.44 (C-2), 87.23 (C-4), 182.11 (C-1); LC-MS m/z: 130.

Compound 3 (2,4-dihydroxy-2,5-dimethylfuran- 3(2H)-one), pale greenish crystals, molecular formula: C₆H₈O₄; yield: 200 mg; R_f: 0.4 (hexane:ethyl acetate, 1:4); melting point: 163-164°; ¹H NMR (CDCl₃, 400 MHz) δ 1.87 (3H, s, 6-CH₃), 2.05 (3H, s, 5-CH₃), 3.62 (1H, s, 3-OH), 3.92 (1H, s, 1-OH); ¹³C NMR (CDCl₃, 400 MHz) δ 12.14 (C-6), 22.41 (C-5), 99.46 (C-1), 139.32 (C-3), 159.75 (C-4), 192.74 (C-2); CHNS analysis: found C-50.00, H-5.60 (%), calcd. C, 50.46, H, 5.54 (%); LC-MS m/z: 144.

Gas chromatography-mass spectrometry (GC-MS) analysis:

The phytochemical investigation of ethanol extract of C. baltica was performed on a GC-MS system (GCMSQP2010 Plus, Shimadzu, Europe). Experimental parameters set for the GC-MS were, column oven temperature- 50°; injection temperature- 250°; injection mode- split; flow control mode- linear velocity; pressure- 29.7 kPa; total flow- 7.3 ml/min; column flow- 0.72 ml/min; linear velocity- 30.7 cm/s; purge flow- 3.0 ml/min; split ratio- 5.0; oven temperature program- 50° with rate of increase of 10°/min, 50-300° with rate of increase of 7.5°/min, hold time of 2.0 min; equilibrium time- 1.0 min; ion source temperature- 210°; interface temperature- 250°; solvent cut time- 3.0 min; detector gain- 0.99+0.20 kV; threshold- 1000; start time- 4.0 min; end time- 37.30 min; ACQ modescan; event time- 0.50 s; scan speep- 2500; start m/z- 34.0 and end m/z- 1090.00.

Cytotoxicity assay:

MCF-7 (breast), DLD-1 (colon), HeLa (cervical), FADU (head and neck), A549 (lung), SKOV3 (ovary) and normal human mammary epithelial (NHME) were kindly provided by National Centre for Cell Science, Pune. The cancer cells were maintained in minimum essential medium (MEM), containing 10 % fetal calf serum (FBS), 5 % mixture of 100 units penicillin and 100 μg/ml streptomycin in presence of 5 % carbon dioxide in an incubator with 90 % humidity at 37° for 72 h.

Cancer cell lines were maintained in MEM (adjusted to 10 % FBS, 1.5 g/ml NaHCO₃, 0.1 mM MEM non-essential amino acids and 1 mM sodium pyruvate). Three days prior to performing assay, the cells were washed with sterilized phosphate-buffered saline and grown using MEM media (supplemented with 0.25 % trypsin in versene-EDTA and 10 % FBS) and mixed to obtain homogeneous suspension of cells. The suspension was taken in a sterilized polypropylene tube and the cell count in each well was determined in a hemacytometer chamber under a microscope using 0.4 % trypan blue solution. The minimal seed density must be 1.9×10⁴ cells per well. All crude extracts and the standard were dissolved in dimethyl sulfoxide (DMSO) to 100 mg/ml and 10 μg/ml, respectively. Doxorubicin and DMSO were used as a standard and control, respectively.

The sulforhodamine B (SRB) assay[13] is based on the estimation of cellular protein content. The prepared samples were taken in 96-well tissue-culture plate and added 190 μl screened ideal cell suspension and mixed occasionally and incubate at 37° with 5 % CO₂ and 90 % relative humidity for 3 h. Then 100 μl cold TCA was added to each well and incubated at 4° for 1 h. After that the plates were gently washed using water, dried using blow dryer and air-dried at room temperature. To each completely dried well, 100 μl of 0.057 % SRB solution was added, kept aside for 30 min and quickly rinse with 1 % acetic acid. To the dried plate, 200 μl of 10 mM Tris base (pH 10.5) solution was added, shake for 5 min and measure the OD at 510 nm. The blank contains only medium while the control has only cancer cells with no test samples. Percent growth inhibition was calculated using the formula, growth inhibition (%) = 100–[(S–B)/(C–B)]×100, where S is mean OD value of sample, B is mean OD value of blank, C is mean OD value of control.

Results and Discussion

After the successful extraction of the whole marine algae material with ethanol-water, the dried ethanol extract was subjected to GC-MS analysis. The GCMS chromatogram revealed the presence of various chemical components with different retention times, whereas the MS analyzes the compounds eluted at different times to detect its nature and structure of the compounds. The results pertaining to GC-MS analysis of the ethanol extract lead to the identification of a number of compounds. A total of 86 compounds were identified by GC-MS analysis of ethanol extract, which were tabulated in Table 1.

S. No	Compound name	Chemical structure	Molecular formula
1	(2Z)-1,3-dimethyl-6-oxo-2-hexenyl acetate		C10H16O3
2	(2Z)-5-methyl-2-decene		C11H12
3	(9E)-9-octadecenal		C18H34O
4	(9Z)-9-octadecenamide		C18H35NO
5	1-((E)-[(2-methylphenyl)imino]methyl)-2-naphthol		C18H15NO
6	1,2,5-trimethyl-4-piperidinone thiosemicarbazone		C9H18N4S
7	1,3-cyclohexanedione		C6H8O2
8	1,4-dimethyl-5-oxabicyclo[2.1.0]pentane		C6H10O
9	1,5-anhydro-6-deoxyhexo-2,3-diulose		C7H10O3
10	10-undecen-1-ylester		C17H32O2
11	1-chloro-4-decyne		C10H17Cl
12	1-hexadecylpyridinium chloride monohydrate		C21H40ClNO
13	1-Tetradecanol, acetate		C16H32O2
14	1-undecene		C11H22
15	3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one		C6H10O3
16	2-(benzyloxy)-4-bromo-1,3-butanediol		C11H15BrO3
17	2,10-dimethylundecane		C13H28
18	2,2,4-trimethyl-1-pentanol		C8H18O
19	2,2-dimethyl-1-phenyl-1-propanol		C11H16O
20	2,2-dioctyl-1-oxohydrazine-1-oxide		C16H34H2O2
21	9,12-Octadecadienoic acid (Z,Z)-, 2,3-bis(acetyloxy)propyl ester		C25H42O6
22	2,3-dihexyl-2-cyclopropene-1-carboxylic acid		C16H28O2
23	2,4,6-trimethyldecane		C13H28
24	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furanone		C6H8O4
25	2,5-furandione		C4H2O3
26	2-acetoxytetradecane		C16H32O2
27	2-acetoxytridecane		C15H30O2
28	2-cyclohexen-1-one		C6H8O
29	2-cyclohexylethyl acetate		C10H18O2
30	2-hydroxy-3-methylbutanoic acid		C5H10O3
31	2-propylheptan-1-ol		C10H22O
32	3-(methoxymethoxy)-butanoic acid		C6H12O4
33	3,5,5-trimethyl-4-(3-oxo-1-butenyl)-2-cyclohexen-1-one		C13H18O2
34	3,5-bis(1,1-dimethylethyl)-phenol		C14H22O
35	3,6-dimethyl-2-octanone		C10H20O
36	3-acetoxydodecane		C14H28O2
37	3-ethyl-2,5-dimethylhexane		C10H22
38	3-hexene-2,5-diol		C6H12O2
39	3-hydroxy-2,2,6-trimethylcyclohexyl-methyl acetate		C12H22O3
40	3-hydroxydodecanoic acid		C12H24O3
41	3-nonyn-1-ol		C9H16O
42	4-acetoxypentadecane		C17H34O2
43	4-heptanol		C7H16O
44	5-(1-benzyl-1h-indol-3-ylmethylene)-1-(2-ethyl-phenyl)-pyrimidine-2,4,6-trione		C28H23N3O3
45	5-bromo-3-methylpentyl acetate		C8H15BrO2
46	5-cyclopropylidene-1-pentanol		C8H14O
47	7,9-ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione		C17H24O3
48	9-octadecenamide		C18H35NO
49	9-octadecenoic acid		C18H34O2
50	Acetic acid		C2H4O2
52	Araldite		C16H22O4
53	Benzenemethanol		C7H8O
54	Benzoic acid		C7H6O2
55	Butanoic acid		C4H8O2
56	Cis-oleic acid		C18H34O2
57	Cyclopentaneundecanoic acid		C16H30O2
58	Dimethyl(ethenyl)propoxysilane		C7H16OSi
59	Di-n-butyl sulphite		C8H18O3S
60	Di-n-octyl phthalate		C24H38O4
61	DL-alanine		C3H7NO2
62	E-2-undecen-1-ol		C11H22O
63	Ethyl-(4E)-2-acetyl-2-methyl-4-hexenoate		C11H18O3
64	Ethyl docosanoate		C24H48O2
65	Ethyl nonadecanoate		C21H42O2
66	Hexadecyl acetate		C18H36O2
67	Methyl-(E)-docos-13-enoate		C23H44O2
68	Methyl-(9E)-9-octadecenoate		C19H36O2
69	Methyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate		C18H28O3
70	Methyl-3-isopropyl-6-oxoheptanoate		C11H20O3
71	Methyl-4-hydroxy-3-methyl-2-butenoate		C6H10O3
72	Methyl heptanoate		C8H16O2
73	n-cyclohexyl methacrylamide		C10H17NO
74	n-decyl acetate		C12H24O2
75	n-heptadecane		C17H36
76	Octadecane		C18H38
77	Octadecanoic acid		C18H38O2
78	Octanal dimethyl acetal		C10H22O2
79	Palmitic acid		C16H32O2
80	Palmitic acid ethyl ester		C18H36O2
81	Pelargonic acid methyl ester		C10H20O2
82	Pentanoic acid		C5H10O2
83	Phthalic acid		C8H6O4
84	Tert-nonanethiol		C9H20S
85	Tetradecane		C14H30
86	Xanthosine		C10H12N4O6

Table 1: Chemical constitutents identified by GC-MS analysis of ethanol extracts of Chara baltica

The collected seaweed was extracted with ethanol water at room temperature. The obtained ethanol extract C. baltica was subjected to chromatographic purification led to isolation of three metabolites (1-3, fig. 1). Compound 1 was obtained as a colorless crystals with R_f value 0.7 (hexane:ethyl acetate, 1:9) and the LC-MS ion peak at m/z 100 confirmed the molecular formula as C₄H₄O₃. The ¹H and ¹³C NMR data showed the presence of two methane groups at δ_H 4.00 (s, 4H) with corresponding carbon signal at δC 22.16 (C-2 and C-3). In addition, the ¹³C NMR also revealed the presence of dione groups at δ_C 170.24 (C-1 and C-4). Hence, compound 1 was confirmed after corroboration with the existing literature[14] as a dihydrofuran-2,5-dione (fig. 1).

Figure 1: Known secondary metabolites from ethanol extracts of Chara baltica

Compound 2 was obtained as a pale greenish liquid. Based on the LC-MS ion peak at m/z 130 confirmed the molecular formula as C₆H₁₀O₃. The 1H NMR showed the presence of two singlets for two methyl groups at δ_H 1.00 (s, 6H) with corresponding carbon signal at δ_C 23.15 (C-5 and C-6), including one methine group at δ_H 2.31 (s, 1H) with corresponding carbon signal at δ_C 80.44 (C-2), and one hydroxyl group at δ_H 4.11 (s, 1H), additionally, two doublets at δ_H 3.86-3.88 (d, J=8 Hz, 1H) and 4.04-4.06 (d, J=8 Hz, 1H), which confirmed the presence of methane group at δ_C 87.23. In addition, the ¹³C NMR also revealed the presence of dione group at δ_C 182.11 (C-1). By corroboration with the existing literature compound 2 was found to be 3-hydroxy-4,4- dimethyldihydrofuran-2(3H)-one (fig. 1)[15].

Compound 3 was obtained as a pale greenish crystals, based on the LC-MS ion peak at m/z 144 confirmed the molecular formula as C₆H₈O₄. The ¹H NMR showed the presence of four singlets for two methyl groups at δ_H 1.87 (s, 3H) and 2.05 (s, 3H), and for two hydroxyl groups at δH 3.62 (s, 1H) and 3.92 (s, 1H). The ¹³C NMR revealed the presence of two methyl group at δ_C 12.14 (C-6) and 22.41 (C-5), two hydroxyl groups at δ_C 99.46 (C-1) and 139.32 (C-3), dione group at δ_C 192.74 (C-2), and one methene carbon signal at δ_C 159.75 (C-4). Based on the aforementioned data, compound 3 was compared with the existing literature on lichen metabolites and it was confirmed as a 2,4-dihydroxy- 2,5-dimethylfuran-3(2H)-one (fig. 1). Compound 3 was earlier reported from Sysepalum dulcificum[16].

Earlier reports of C. baltica indicated free radical scavenging activity[13]. Based on the report, the isolated metabolites (1-3) were screened along with ethanol extract against six different cancer cell lines using SRB assay with doxorubicin as standard. Initially, compounds (1-3) at 30 μg/ml concentration, extract at 100 μg/ml concentration and doxorubicin at 10 μg/ml concentration were screened against the cell lines tested and percent inhibition of cell growth was tabulated in Table 2.

Table 2: Cytotoxicity studies of compounds 1-3 and ethanol extract of Chara baltica

Further, the samples that caused 50 % or more of cell death were further screened at 5, 10, 20 and 30 μg/ml concentrations for pure compounds; 25, 50, 75 and 100 μg/ml concentrations for extracts; 2.5, 5.0, 7.5 and 10 μg/ml concentrations for doxorubicin. The results were plotted to obtain IC₅₀ values. The lower IC₅₀ value indicated better inhibitory profile against cancer cell lines.

During the initial screening, the isolate 3 and ethanol extract depicted prominent degree of specificity against MCF-7, DLD-1, FADU and A549. Besides, all the samples exhibited very low toxicity towards NHME cell lines (Table 2). From the outcomes of the SRB assay, it is interesting to note that compound 3 (30 μg/ml) and ethanol extract (100 μg/ml) showed prominent degree of specificity against DLD-1 with 76.37±5.34 and 87.11±3.58 % growth inhibition which was found to be better than activity of 66.71±0.71 % shown by the standard at 10 μg/ml (Table 2).

From the Table 3, the IC₅₀ values of 3 and ethanol extract against MCF-7 were 26.5 and 75.0 μg/ml, respectively, whereas standard value was 5.5 μg/ml. In addition, the concentration of 3 and ethanol extract needed for 50 % inhibition of DLD-1 were found to be 15.5 and 61.0 μg/ml, respectively, while for doxorubicin it was 5.4 μg/ml (Table 3).

Table 3: Growth inhibition and IC₅₀ values of compound 3 andethanol extracts of Chara baltica

From the SRB assay of FADU (Table 3) it can be calculated that the IC₅₀ values of 3 and ethanol extract against FADU were 16.5 and 74.5 μg/ml, respectively, whereas standard value was 4.5 μg/ml. Additionally, the concentration of ethanol extract needed for 50 % inhibition of A549 was found to be 74.5 μg/ml, while standard (doxorubicin) was 4.5 μg/ml (Table 3).

Hence, from the overall SRB assay it can be concluded that the key agent responsible for anticancer activity is compound 3. In addition, the growth inhibitory properties of compound 3 is mainly due to the higher levels of oxygenated substituents present in its chemical structure, this higher levels of oxygenated content help in irradiating free radicals that are usually formed during massive cell division of cancer cells, which ultimately lead to the cell death[13,17].

In conclusion, this is the first report of GC-MS analysis, chemical and cytotoxicity profile of marine algae C. baltica. The GC-MS analysis of ethanol extract C. baltica revealed presence of 89 chemical constituents. In addition, the chemical examination of ethanol extract showed the presence of three metabolites 1-3. Additionally, the cytotoxicity studies of ethanol extract indicated specificity towards MCF-7, DLD-1, FADU and A549 without affecting NHME. The SRB assay screening proved that compound 3 has greater cytotoxicity. The results of the current study could be useful for further research on cancer to identify potential bioactive molecules from different aquatic fauna, such as marine algae like C. baltica.

Acknowledgement:

The authors thank the authorities of CDRI-Lucknow, India for providing the cytotoxicity testing facilities to complete present work. The authors wish to thank Dr. G. Mohan Narasimha Rao, Professor of Botany Department, Andhra University, Visakhapatnam, for authenticating the seaweed.

Conflict of interest:

No conflict of interest between any of the authors.

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