The GC-MS chromatogram of ethyl acetate extract from P. granatum seed is shown in Fig. 1. The GC-MS analysis of ethyl acetate extract from P. granatum (Table 1) led to the identification of twelve major compounds out of which one compound is an alkene namely 1-Methoxy-2-methyl-3-butene (11.23%), one compound is an alcohol(13.36%) 1-butanol, four compounds are aldehydes 3-methyl, 2- hexenal, 2,4-Nonadienal, 2,4-Decadienal, 5-hydroxy methyl furfural which amounts to 22.97%, one compound is a heterocyclic compound Methyl-2- phenylindole (5.86%), and five compounds are fatty acids Tetradecanoic acid, Hexadecanoic acid, linoleic acid, linoleic acid, Octadecanoic acid and Octadecatrienoic acid (43.53%). This constitutes about 91.95% of the total constituents (Panighel et al., 2014); Pinto et al., 2019). Fatty acids dominate among the compounds of the extract. Among all the compounds present in the extract the major compound is Octadecatrienoic acid which constitutes 19.88% of the whole extract. It was followed by linoleic acid(16.78%).
Table 1
GC-MS Phyto constituents of crude extract from ethyl acetate Punica granatum seed.
S.NO | Name | RI | Area % | Formula | Exact Mass |
1 | 1-Methoxy-2-methyl-3-butene | 624 | 11.23 | C6H12O | 100.08 |
2 | 1-Butanol, 3-methyl- | 733 | 13.36 | C6H10O2 | 100.16 |
3 | 2- hexenal | 820 | 10.58 | C6H12O6 | 100.13 |
4 | 2,4-Nonadienal, | 854 | 3.40 | C9H14O | 138.10 |
5 | 2,4-Decadienal, (E,E)- | 1256 | 3.34 | C10H16O | 152.12 |
6 | 5-hydroxy methylfurfural | 1267 | 5.65 | C6H6O3 | 126.12 |
7 | Methyl-2- phenylindole | 1665 | 5.86 | C15H13N | 207.27 |
8 | Tetradecanoic acid | 1730 | 1.75 | C14H28O2 | 228.34 |
9 | Hexadecanoic acid | 1984 | 1.69 | C16H32O2 | 256.42 |
10 | linoleic acid | 2078 | 16.78 | C13H26O | 226.17 |
11 | Octadecanoic acid | 2161 | 3.43 | C18H34O2 | 282.5 |
12 | Octadecatrienoic acid | 2207 | 19.88 | C18H30O2 | 88.11 |
Isolation And Characterization Of Pg-1
The P.granatum fruit was cut in to small pieces and after the removal of the exocarp and mesocarp portions, the seeds were collected. After the removal of the juicy liquid portion from the seeds they were shade dried, grounded to powder and extracted exhaustively with ethyl acetate using a Soxhlet apparatus. The extract was concentrated and subjected to column chromatography to isolate PG 1. Elution of the ethyl extract with a mixture of petroleum ether: ethyl acetate ( 8:2) yielded a yellow colored (440 mg) PG -1. It was characterized and the structure was identified using its UV, IR, NMR and MS data.
The LC-MS positive mode spectra of PG-1 showed a pseudo molecular ion [M + H]+ peak at 916.34 indicating a molecular weight of 915.42 and analyzed for a molecular formula; C60 H98 O6. In the 1H NMR spectrum, the triplet signals at δ 0.88, 0.90, and 0.92 for nine protons are attributed to three terminal methyl groups. It was complemented by the appearance of the corresponding carbon atoms resonated at δ 13.93, 13.96, and 14.12 respectively in the 13C-NMR spectra (Aung et al., 2017). The bulky methylene group protons appeared at δ 1.25 and 1.30 indicated the presence of fatty acid moieties. The pair of a doublet of doublets signals at δ 4.26 and 4.34 is assigned to the acylated oxymethylene protons of the glycerol moiety whereas the acylated methylene proton resonated at δ 5.26, thereby suggesting that PG-1 is a triglyceride fatty ester. The 13C-NMR signals which strongly support the triglyceride structure for PG-1 is presented in the Table 2.
Table 2
The 13C-NMR signals of the PG-1
Carbon | Signal (δ) |
Glycerol Moiety | |
1—CH2OCOR | 62.11 |
2- CHOCOR | 68.89 |
3- CH2OCOR | 62.11 |
Fatty acid | |
-C = O | 173.27 |
| 172.86 |
| 173.27 |
-CH = CH- | 135.23 |
| 134.52 |
| 134.26 |
| 132.88 |
| 132.71 |
| 132.48 |
| 132.03 |
| 131.78 |
| 130.90 |
| 130.75 |
| 130.54 |
| 130.42 |
| 128.73 |
| 128.63 |
| 128.09 |
| 127.80 |
| 126.10 |
| 125.94 |
-CH2CO- | 34.19 |
| 34.03 |
| 32.76 |
-CH2-CH = CH-CH2 | 31.93 |
| 31.52 |
-CH = CH-CH2-CH = CH | 29.71 |
- CH2CH2CO | 29.53 |
-CH2- | 29.32 |
| 29.13 |
| 29.00 |
| 27.81 |
| 27.55–22.23 |
- CH3 | 14.12 |
| 13.96 |
| 13.93 |
The multiple signal at δ 5.60 and 6.02 in the 1H NMR spectra suggests the presence of many unsaturated -CH = CH- bonds. It was complemented by its 13C-NMR spectra by exhibiting eight signals between δ 126.10 and 134.52 (Zhong et al., 2017). The signals at δ 2.84 in 1H NMR for two protons are assigned to protons attached to bis-allylic carbon atoms of the fatty acid (Fig S1). The allylic protons resonated at δ 2.07 and the protons attached to the α-carbon atoms of the fatty acid moiety appeared at δ 2.31. The protons attached to the β- carbon atoms appeared at δ 1.60. The two signals one signal with double the intensity, corresponds to three carbonyl functions at δ 173.27, 172.86, terminal methyl groups three signals at δ 14.12, 13.92, and 13.93, a glyceridic primary carbon atom at 62.12 in the 13C-NMR spectra (Fig S2), and the terminal methyl groups at δ 0.89 in the 1H-NMR in the same environment suggests that the three acids may be the same and are esterified in the oxymethylene group of the glycerol moiety. The acid appears to be punic acid. All the other signals as indicated in Table 2 indicated that the compound is triacylated glycerol. Based on the above spectral data the compound was identified as tri-O –punicyl glycerol (Fig. 2).
Uv-vis And Ft Ir Spectral Analysis
UV-visible spectra analysis showed an absorption maximum λmax at 250 nm (Fig S3). FT-IR spectra of PG-1 showed characteristic absorption bands at 1740 cm-1 for (C = O) Carbonyl group, at 1640 cm-1for C = C group and at 1100 cm-1 for C-O group (Fig S4).
In vitro antioxidant activity of Compound 1
The radical scavenging activity of PG-1 was evaluated by DPPH assay and ABTS assay methods. In DPPH assay the IC50 value of PG-1 and the standard compound L. Ascorbic acid were 26 ± 5.7 µg/ml, 24 ± 6.8 µg/ml. respectively as shown in Fig. 3. Evaluation of the antioxidant capacity by the ABTS assay method showed an IC50 value 26 ± 0.62 µg/ml and 23 ± 0.65µg/ml, for PG-1 and L-ascorbic acid (Fig. 4) respectively. The compound possess a comparable scavenging activity to the standard compound (Mandal et al., 2015) in both the methods.
Antiproliferative Activity Of Pg-1
MTT assay
The in vitro cytotoxic activity of PG-1 was determined against A549 lung cancer cell line by MTT assay (Mosmann et al., 1983). The anticancer properties showed that the PG-1 had no toxicity on fibroblast cell line (3T3) whereas on lung cancer cell line, PG-1 inhibited the growth of cancer cells and the IC50 value, inhibition concentration was 25 ± 8.5µg/ml and that of L- ascorbic acid was 22 ± 7.4 as shown in Fig. 5–7 (Nelson et al., 2019; Khayam et al., 2020).
Genotoxicity Activity Of Pg-1 On Lung Cancer Cell Line By Comet Assay
The results of Genotoxicity of PG-1 on lung cancer cell line was analyzed using comet assay (Mei et al., 2010) and DNA damage was observed in A549 cell line at the concentration of 250µg/ml. Using an open comet software, an increase in the % tail DNA damage was observed as compared to control. Longer the tail, more is the damage (Rad et al., 2017) and PG-1 was capable of destroying cancer DNA. thereby it signified that the PG-1 behaves as an effective anticancer candidate Figs. 8 and 9 (Ramos et al., 2016).
Cell cycle Analysis of PG-1 on lung cancer cell line by flow cytometer.
The cell cycle distribution analysis using flow cytometer was carried out for the PG-1 to find out its effect on A549 cell line. After 48 hours, at 12µg/ml concentration it indicated that the cells were arrested at G0 phase as compared to control (Modem et al., 2012; Li et al., 2016). The treated A549 Cell line with PG-1 was blocked in G0 cell cycle phase (Fig. 10). At time 0, most cells (60%) were accumulated, due to the high proliferative state of this cell line. Untreated control cells showed the expected pattern for continuously growing cells. Hence this shows PG-1 is effective in cell cycle arrest and thereby qualifies for drug design and clinical trials for lung cancer (Siddiqui et al., 2014).
Suggesting a triglyceride as a lead molecule for cancer therapy may seem rather contrary to common-sense expectation. We may think that the presence of triglycerides in the blood at higher levels is a risk for cancer initiation and progression, because they may act as a rich source of energy for the developing tumors (Ulmer et al., 2009; Shen) et al., 2018). Nevertheless, for few types of cancers like breast cancer (Ni et al., 2015), prostate cancer (Ulmer et al., 2009) high levels of circulating triglycerides correlate with a better prognosis. Further in few studies, it was reported that specific lipids like linoleic acid has been suggested as a natural anticarcinogenic compound (Cannella and Giusti, 2000; Marcos. Et al., 2020).
Further worldwide, Lung cancer is considered as one of the leading causes of death in both men and women and cigarette smoking is attributed to the major cause of the condition, because cigarette smoke causes an increase in oxidative stress and DNA damage. In the present study since PG-1 possess both anticytotoxic activity and antioxidant activity this can act as a natural plant based medicine with lower side effects against lung cancer cell line (A549). Also it can be used as an adjuvant as well in the therapy of lung cancer for the patients using methotrexate for their cancer therapy.