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. 2017 May 12;7(1):85. doi: 10.1007/s13205-017-0678-9

Phytochemical profile and free radical nitric oxide (NO) scavenging activity of Averrhoa bilimbi L. fruit extract

Jagadish Kumar Suluvoy 1, V M Berlin Grace 1,
PMCID: PMC5429310  PMID: 28500407

Abstract

Averrhoa bilimbi L. belongs to family Oxalidaceae. Traditionally, people use this plant (root, bark, leaves and fruits) for treating several illnesses include itches, boils, syphilis, whooping cough, hypertension, fever and inflammation. The aim of the study was to evaluate the nitric oxide (NO) scavenging activity and GC–MS analysis of A. bilimbi L. fruit extract. Averrhoa bilimbi L. fruits were collected for the preliminary phytochemical analysis, antioxidant scavenging activity and biologically important compounds were identified by GC–MS analysis. The preliminary phytochemicals, GC–MS, total phenolic content and NO scavenging activity of the plant were analysed. In the present investigation, the A. bilimbi L. fruit extract has major phytochemicals. Among the 151 compounds identified in GC–MS, 15 compounds are found to have diverse biological activity. We also observed that the A. bilimbi L. fruit extract has high level of total phenolic compounds at a concentration of 209.25 GAE mg/g. Presence of phenolic compound apparently explains the antioxidant activity of the plant. Antioxidant activity of A. bilimbi L. fruit extract is proven from its high level of NO scavenging activity of potent IC50 value of 108.10. From the above study, it is apparent that the A. bilimbi L. fruit extract is a rich source of phytochemicals (natural products) with biological activity. The GC–MS report on this fruit proves that natural products have pharmacologically and biologically active compounds. A high phenolic content is observed in our study. A. bilimbi L. fruit extract is also found to have NO scavenging activity in our study.

Keywords: Averrhoa bilimbi L., GC–MS, Nitric oxide, Phenol, Phytochemical, Antioxidant

Introduction

Everyday 50,000 premature deaths are caused due to infectious diseases (Singh et al. 1992; Robin et al. 1998). In accordance with the World Health Organization (WHO) 2014 diseases like malaria, dengue, leishmaniasis, Lyme disease, tuberculosis, schistosomiasis, and yellow fever, carried by mosquitoes, flies, ticks, water snails and air infect one billion people and more than one million people will die. Pathogens and diseases become drug resistant and the best alternate approach are plants to eliminate diseases and therapeutic complications (Fabricant and Farnsworth 2001). From time immemorial plants are used as medicine to treat diseases. Before the discovery of allopathy humans depended on Ayurveda and homeopathy medicine which are completely based on plants and herbs. These herbs and plant materials act as medicine to cure diseases (Nostro et al. 2000). Tribal people depend on the rich diversity of forest to overcome the health care needs. Forests have excellent vegetation (flora) with high quality of medicinal value (Kadhirvel et al. 2010). Phytochemicals are the non-nutrient compounds with beneficial health effects leading to pharmacological importance and are used in medication (Nisa et al. 2011). Fruits play major role in human diet due to their bioactive compounds, natural sugars and organic acids with relatively high antioxidant activity (Rechkemmer 2001) and are a rich source of vitamins (A, B6, C, E, niacin, and thiamine) dietary fibre and minerals (Wargovich 2000). Averrhoa bilimbi L. is a long-lived green plant which gives edible fruits, belonging to the family Oxalidaceae–Oxalis and grows 16–33ft (5–10 m) in height, with short trunk dividing into number of upright branches. It is found throughout Malaysia, Indonesia, Myanmar, Bangladesh, Srilanka and common in Southeast Asian countries (Rahman et al. 2014). In India it is available mostly in Kerala regions, particularly the Kani tribal traditional healers in Thodu hills region (Kerala) use the raw leaves and fruits of A. bilimbi L. plant for ailments in circulatory system (Xavier et al. 2014) and the local name is Irumban puli or Pulingi. The parts like bark, leaves, seeds, flowers, fruits, roots and the entire A. bilimbi L. plant is used as alternative medicine to treat numerous diseases majorly as anti-diabetic agent (Kumar et al. 2013). Traditionally it is used in medication to cure cough, cold, boils, itches, syphilis, whooping cough, rheumatism and hypertension (Sabiha et al. 2012). A. bilimbi L. shows antimicrobial activity against gram positive and gram negative bacteria (Karon et al. 2011), antifungal activity (Nazmul et al. 2011), cytotoxic activity (Das et al. 2011), anti-diabetic activity (Pushparaj et al. 2000) and the leaves of A. bilimbi L. could increase the serum insulin level (Patel et al. 2012) in diabetes mellitus. Administration of A. bilimbi L. fruit (toxicity studies) extract 1 g/kg bw did not affect the mice (Savithri et al. 2009). However, in spite of having of such a great traditional medicinal use, the knowledge on its phytochemical is limited. Few studies of phytochemicals on the A. bilimbi L. fruit extract have shown contradictory data on the presence of alkaloids, tannins, glycosides, saponins and steroids (Sabiha et al. 2012). This study is therefore designed to analyse the active phytochemicals present in the fruits of A. bilimbi L. by biochemical tests and GC–MS which may be useful for exploring its ethno-pharmacological significance and to validate scientifically its medicinal properties. Several previous studies have shown the free radical scavenging activity of A. bilimbi L. fruit extract through DPPH scavenging activity (Asna and Noriham 2014; Sabiha et al. 2012). Oxidative stress has major action in human anatomy, physiology and diseases like cardiovascular diseases, diabetes, inflammatory conditions, ageing and cancer (Joyce 1987). Nitric oxide (NO) plays a major role in several in vivo diseases like neuronal signalling, smooth muscle relaxation, regulation of cell-mediated toxicity and inhibition of platelet aggregation (Hagerman et al. 1998). Surplus NO is reported to direct DNA fragmentation, cell damage and neuronal cell death. NO will not affect the DNA and proteins directly but NO is very unstable in aerobic condition and produces NO2, N3O4, N2O4 intermediates which are genotoxic, affecting the DNA repair proteins and also deaminate DNA bases (Umamaheswari and Chatterjee 2008). Hence it is essential to reduce the levels of NO in human body. Besides to the reactive oxygen species (ROS) NO was found to be elevated in inflammation (Baygutalp et al. 2015), colon cancer (Erdman et al. 2009) and pathological conditions like gastrointestinal disorders (Cho 2001). The traditional usage of the fruits of A. bilimbi L. for anti-inflammation, anti-diabetic and anti-hypertensive was highlighted in a report on Malaysian medicinal plants (Harun et al. 2015). A. bilimbi L. is a rich source of vitamin C, A, B1 and 100 g of edible portion was found to have moisture, 94.2–94.7 g; fibre, 0.6 g; ash, 0.31–0.40; protein, 0.61 g; calcium, 3.4 g; iron, 1.01 mg; riboflavin, 0.32 mg; thiamine, 0.010 mg; ascorbic acid, 15.5 mg (Zakaria et al. 2007). Ascorbic acid has been used as standard drug for the estimation of nitric oxide scavenging activity (singh et al. 2012). As the A. bilimbi L. fruit extract has also shown a good antioxidant potential against DPPH (Chauhan and Kapfo 2013), we made an attempt to analyse its in vitro NO scavenging activity.

Materials and methods

Chemicals and reagents

All the chemicals used for this experiment were analytical grade purchased from Hi-Media, and SD Fine Chemicals.

Selection and authentication of fruit

Averrhoa bilimbi L. fruit samples were collected from Palakkad district, Kerala, during February to March (2015). The fruits of A. bilimbi L. are used as a source of food and medicine by tribes and settler communities of the local people. The authentication of the fruit was done by the Botanical Survey of India (BSI) Coimbatore, Tamilnadu, India. The authentication number given by the BSI is BSI/SRC/5/23/2015/Tech.

Extraction of fruit material

The fruits of the A. bilimbi L. were collected, air dried and made into fine powder by the mortar and pestle. Extraction from the fruits was done according to the method described by Singh et al. (2012). The powder (25 g) was used for the extraction with 250 ml of methanol (95% v/v) in a soxhlet apparatus. The remaining methanol was evaporated using rotary evaporator. The obtained thick semi-solid crude extract was stored at 2–4 °C for further use.

Phytochemical screening of A. bilimbi L. fruits

The A. bilimbi L. fruit (methanol) extract was analysed for the presence of alkaloid, carbohydrate, glycosides, phenols, flavonoids, saponins, steroids and tannins using the respective biochemical tests as follows.

Test for alkaloids

Two millilitre of 1% HCl was mixed with 0.1 gm of crude extract and heated slightly. After cooling Wagner’s reagent and Mayer’s reagent were added to it. The presence of buff-coloured precipitate indicated the presence of alkaloids (Sofowora 1993).

Test for carbohydrates

Benedict’s reagents was mixed with the 0.1 gm of crude extract and slightly boiled, appearance of reddish brown precipitate indicated the presence of the carbohydrates (Harborne 1973).

Test for flavonoids

The appearance of pink scarlet colour when 0.1 gm of crude extract was mixed with few drops of concentrated HCl and Mg pellets indicated the presence of flavonoids (Odebiyi and Sofowora 1978).

Test for phenols

Two millilitre of 2% ferric chloride was mixed with the 0.1 gm of crude extract and the presence of blue-green or black coloration indicated the presence of phenols (Yadav and Agarwala 2011).

Test for saponins

Saponin presence was detected by the frothing test. Briefly 0.1 gm of crude extract was mixed well in water and shaken, the appearance of foam indicated the preliminary evidence for the presence of saponins (Kumar et al. 2009).

Test for steroid (Liebermann test)

0.1 gm of crude extract was mixed with 2 ml H2SO4 and slowly added to 2 ml of acetic anhydride. The colour change from violet to green or blue indicated the presence of steroids (Edeoga et al. 2005).

Test for tannins

0.1gm of crude extract of A. bilimbi L. fruit was mixed in distilled water and filtered. Few drops of ferric chloride solution were added to the filtrate. The green or blue-green precipitate indicated the presence of tannins (Trease and Evans 2002).

GC–MS analysis on A. bilimbi L. fruit extract

Averrhoa bilimbi L. methanolic fruit extract was subjected to gas chromatography–mass spectroscopy (GC–MS) analysis. The Thermo GC-Trace Ultra VER: 5.0 (Bremen, Germany) and Mass Spectroscopy (MS) MS DSQ II electron ionization mode with ionization energy of 70 eV were used. The temperature of the column was set to 80–250 °C at 8 °C/min rate. Temperature of 280 and 290 °C were set for the GC injector and MS transfer, respectively. Helium was used as a carrier gas at a flow rate of 1.0 ml/min. The sample volume of 1 μl was used for analysis. By the retention time and mass fragmentation patterns, the major compounds present in the fruit extract were analysed. The National Institute of Standards and Technology (NIST) and Wiley 9.0 library was used (Sakthivel and Guruvayoorappan 2013) for the detection of compounds.

Estimation of phenols in A. bilimbi L. fruit extract

The total phenolic compounds were estimated using the Folin–Ciocalteu reagent (Slinkard and Singleton 1977). Briefly 0.1 ml of A. bilimbi L. fruit extract of different concentrations (50, 100, 150, 200, and 250 µg/ml) were mixed with 2 ml of 10% Folin–Ciocalteu reagent and 3 ml of 7% Na2 CO3 was added. This was incubated for 30 min at room temperature and the absorbance was measured using UV-spectrophotometer at 760 nm. Gallic acid was used as standard and all the results were performed in triplicates. The total phenol concentration is expressed in mg gallic acid equivalent (GAE).

Nitric oxide (NO) scavenging activity of A. bilimbi L. fruit extract

The nitric oxide (NO) scavenging activity of the A. bilimbi L. fruit extract was expressed in percentage inhibition (Vaijanathappa et al. 2008). Briefly 3 ml of 10 mM sodium nitroprusside (0.5 mM PBS pH 7.4) was mixed with 1 ml of A. bilimbi L. fruit extract at different concentrations (25, 50, 75, 100, 125, and 150 µg/ml) and incubated at 25 °C for 150 min. Then 0.5 ml of the reaction mixture was removed and 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) was added and again incubated for 5 min at 25 °C. After adding 1 ml of naphthyl ethylene diamine dichloride (0.1 w/v), the entire reaction mixture was allowed to stand for 30 min at room temperature. The absorbance was measured at 540 nm. Similar procedure was repeated for the standard ascorbic acid at different concentrations (25, 50, 75, 100, 125, 150 µg/ml). The same reaction mixture with the methanol served as control (without extract and standard)

%Inhibition=(A0-A1)/A0×100,

where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.

Results

Phytochemical analysis

Preliminary phytochemical tests revealed the presence of alkaloids, carbohydrates, phenols, flavonoids, saponins and tannins (Table 1). The presence of more phenols was observed in the preliminary screening. The test for sterols answered negative in our study.

Table 1.

The phytochemicals present in A. bilimbi L. fruit extract. It reveals the presence of alkaloids, carbohydrates, phenols, flavonoids, saponins, tannins. (++ indicates more amount) and the absence of steroids (−)

S. no Test Results
1 Alkaloids +
2 Carbohydrates +
3 Phenols ++
4 Flavonoids +
5 Saponins +
6 Steroids
7 Tannins +

Phytochemical compounds identified by GC–MS analysis

By comparing with the National Institute of Standards and Technology (NIST) and Wiley 9.0 library, the major compounds are identified and listed in Table 2. The GC–MS chromatogram is shown in Fig. 1. Among the 151 compounds identified, 15 compounds are found to have various biological activity which were reported from other studies as mentioned in Table 3. Furthermore, the GC–MS analysis reported the presence of various phenol, flavonoid, lipid, alkaloid and acid compounds which were shown in basic phytochemical screening test.

Table 2.

Compounds identified by GC–MS in the A. bilimbi L. fruit extract

S. no Compound Empirical formula Empirical weight Probability Area  %
1 N-Methoxy-N-methylacetamide C4H9NO2 103 26.21 2.96
2 Propane nitrile, 3-(methylthio)-(CAS) C4H7NS 101 12.73 2.96
3 d-Mannitol C6H14O6 182 7.33 2.96
4 d-Glycero-d-manno-heptitol C7H16O7 212 4 2.96
5 Propionic acid, 2-mercapto-, allyl ester C6H10O2S 146 3.69 2.96
6 Boronic acid, ethyl-, bis(2-mercaptoethyl ester) C16H15BO2S2 194 3.69 2.96
7 N1-Methyluracil C5H6N2O2 126 13.48 3.26
8 d-alanine, N-propargyloxycarbonyl-, isohexyl ester C13H21NO4 255 7.91 3.26
9 Uracil, 1-n-methyl C5H6N2O2 126 6.06 3.26
10 1,5-Bis(dimethylpiperidyl)-2,2-dimethylpentane C21H42N2 322 5.82 3.26
11 d-alanine, N-propargyloxycarbonyl-, decyl ester C17H29NO4 311 5.14 3.26
12 2,2-Diethyl-N-ethylpyrrolidine C19H33NO4 155 4.74 3.26
13 l-alanine, n-propargyloxycarbonyl-, dodecyl ester C19H33NO4 339 3.63 3.26
14 N-Cyano-3-oxobutanamide C5H6N2O2 126 3.21 3.26
15 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one C6H8O4 144 90.5 2.05
16 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl(CAS) C6H8O4 144 90.5 2.05
17 (2R*, 3R*)-2-butyl-3-hydroxy-3-phenylpropionoic acid ethyl ester C15H22O3 250 0.34 2.05
18 2-n-Propylthiane C8H16S 144 0.26 2.05
19 5-Hydroxymethylfurfural C6H6O3 126 75.99 12.5
20 2-Furancarboxaldehyde, 5-(hydroxymethyl)- (CAS) C6H6O3 126 75.99 12.5
21 5-(hydroxymethyl)-2-Furanecarboxaldehyde C6H6O3 126 20.71 12.5
22 5-Hydroxymethyl-2-furaldehyde C6H6O3 126 75.99 12.5
23 Thienylethanal C6H6OS 126 1.4 12.5
24 5-(hydroxymethyl)-2-(dimethoxymethyl)Furan C8H12O4 172 65.9 2.41
25 Oxiraneethanol, á-(1-ethoxyethoxy)-, [2R-[2R@[r@(R@)]]] C9H14O3 170 8.04 2.41
26 4-Methoxymethoxy-4-methyl-hex-2-ynal C9H14O3 170 8.04 2.41
27 2-(methoxycarbonylmethylidene)-5-Hydroxymethyltetrahydrofuran C8H12O4 172 1.85 2.41
28 4-Hydroxylamino-6-methylpyrimidin-2(1H)-one C5H7N3O2 141 1.56 2.41
29 Methyl 4-chloro-2,2-dimethyl-4-pentenoate C8H13ClO2 176 1.1 2.41
30 3-(hydroxymethyl)-9-Oxabicyclo [3.3.1] nonan-3-ol C9H16O3 172 0.82 2.41
31 Methanal, (5-methyl-3-isoxazolyl)amino-, oxime C5H7N3O2 141 0.72 2.41
32 Ethyl 4-hydroxy-3-methylbut-2-enoate C7H12O3 144 87.65 1.1
33 (E)-ethyl-4-hydroxy-3-methylcrotonate C7H12O3 144 2.38 1.1
34 Acetic acid, 2-methylhex-3-yl ester C9H18O2 158 0.53 1.1
35 2,4,4-Trimethyl-2-pentyl-3-oxa-zolidinyloxy C11H22NO2 200 0.45 1.1
36 4,5-Dihydro-2-methyl-5-(nitrimino)-1H-tetrazole C2H4N6O2 144 0.27 1.1
37 1-Naphthyl n-propyl carbamate C14H15NO2 229 0.26 1.1
38 Glutaric acid, 2,4-dichlorobenzyl hexadecyl ester C28H44Cl2O4 514 0.25 1.1
39 Glutaric acid, 3,4-difluorobenzyl nonyl ester C21H30F2O4 384 0.25 1.1
40 Glutaric acid, 3-heptyl hexyl ester C18H34O4 314 0.22 1.1
41 Glutaric acid, decyl 2,5-difluorobenzyl ester C22H32F2O4 398 0.22 1.1
42 (−)-Hygroline C8H17NO 143 19.21 5.12
43 dl-Proline, 5-oxo-, methyl ester C6H9NO3 143 8.17 5.12
44 (Z)-2-Pentenal C5H8O 84 7.22 5.12
45 Methyl pyroglutamate C6H9NO3 143 6.38 5.12
46 l-Proline, 5-oxo-, methyl ester (CAS C6H9NO3 143 6.38 5.12
47 dl-Proline, 5-oxo-, methyl ester C6H9NO3 143 8.17 5.12
48 (+)-Sedridine [2-(2-hydroxypropyl) piperdine] C8H17NO 143 6.13 5.12
49 rac-5-oxopyrrolidine-2-carbonsaure-methylester C6H9NO3 143 4.45 5.12
50 l-Proline, 5-oxo-, methyl ester C6H9NO3 143 6.38 5.12
51 Cyclohexanone, 2,3,4-trihydroxy-6-methyl-, [2S-(2à,3á,4á,6à)] C7H12O4 160 10.49 1.05
52 Guanosine (CAS) C10H13N5O5 283 7.83 1.05
53 2-Amino-9-(3,4-dihydroxy-5-hydroxymethy l-tetrahydro-furan-2-yl)-3,9-dihydro-puri C10H13N5O5 283 7.22 1.05
54 (2s,3r,4r,6r)-2,3,4-trihydroxy-6-methylcyclohexanon C7H12O4 160 10.49 1.05
55 Xanthosine (CAS) C10H12N4O6 284 5.67 1.05
56 Guanosine (CAS) C10H13N5O5 283 7.83 1.05
57 à-d-Galactopyranoside, methyl 3,6-anhydro- (CAS) C7H12O5 176 5.23 1.05
58 2-Deoxy-d-galactose C6H12O5 164 4.83 1.05
59 d-fructose, 1,3,6-trideoxy-3,6-epithio- (CAS) C6H10O3S 162 3.79 1.05
60 2-Cyclohexylpiperidine C11H21N 167 47.66 0.83
61 à-Pyrrolidone, 5-[3-hydroxybutyl]- C8H15NO2 157 47.66 0.83
62 l-Serine, O-(phenylmethyl)- (CAS) C10H13NO3 195 7.52 0.83
63 2-[p-chlorobenzyl]Piperidine C12H16ClN 209 3.2 0.83
64 Formyl glutamine C9H14N2O5 230 2.95 0.83
65 4-[Dichloromethyl]-2-[[2-[1-methyl-2-pyrrolidinyl]ethyl]a mino-6-trichloromethylpyrimidine C13H17Cl5N4 404 2.26 0.83
66 Tridecanedioic acid (CAS) C13H24O4 244 1.92 0.83
67 à-Methyl-l-sorboside C7H14O6 194 88 3.97
68 Methyl-à-d-fructopyranoside C7H14O6 194 7.92 3.97
69 2-Methylacetophenone-dioxolane C11H14O2 178 0.51 3.97
70 d-glucose (CAS) C6H12O6 180 0.2 3.97
71 á-d-Glucopyranose, 4-O-á-d-galactopyranosyl C12H22O11 342 0.19 3.97
72 Isopropyl-á-d-thiogalactopyranoside C9H18O5S 238 0.15 3.97
73 4′-Methylphenyl-1C-sulfonyl-á-d-galactoside C13H18O7S 318 0.15 3.97
74 Ethyl-1-thio-á-d-glucopyranoside C8H16O5S 224 0.14 3.97
75 Galactopyranodise, 1-deoxy-1-undecylthio C17H34O5S 350 0.1 3.97
76 2-Octenoic acid, 4,5,7-trhydroxy C8H14O5 190 9.79 1.26
77 Desulphosinigrin C10H17NO6S 279 8.65 1.26
78 2-Octenoic acid, 4,5,7-trhydroxy C8H14O5 190 8.32 1.26
79 2-d,2-pentadecyl-1,3-dioxepane C20H39DO2 312 6.7 1.26
80 2-Acetylamino-3-hydroxy-propionic acid C5H9NO4 147 5.4 1.26
81 2-acetylamino-3-hydroxy-propionic acid C5H9NO4 147 4.56 1.26
82 2-Hydroxyhexadecyl butanoate C20H40O3 328 3.58 1.26
83 2-[(N,N-Dimethylamino)methyl]-4-fluorophenol C9H12FNO 169 38.97 0.49
84 1-Isobutyl-7,7-dimethyl-hexahydro-isobenzofuran-3a-ol C14H26O2 226 5.55 0.49
85 Hydrazinecarboxamide, 2-(2-methylcyclohexylidene)- C8H15N3O 169 38.97 0.49
86 1,3-Diethyl-1,3,3a,5,6,6a-hexahydrocyclopenta[c]thiophen -4-one C11H18OS 198 3.92 0.49
87 2-Furoic acid, bromomethyldimethylsilyl ester C8H11BrO3Si 262 3.16 0.49
88 2-Furancarboxylic acid, tert-butyldimethylsilyl ester C11H18O3Si 226 2.35 0.49
89 Hydrazinecarboxamide, 2-(2-methylcyclohexylidene)(CAS) C8H15N3O 169 4.25 0.49
90 3-Furoic acid, benzyldimethylsilyl ester C14H16O3Si 260 2.08 0.49
91 2-Furoic acid, (3-cyanopropyl)dimethylsilyl ester C11H15NO3Si 237 2.93 0.84
92 Chimanine D C12H11NO 185 39.01 0.55
93 Methyl 5-(N-Hydroxy)carboximidamido-2-thiophenecarboxylate C7H8N2O3S 200 29.88 0.55
94 Octadecanoic acid, 2,3-dihydroxypropyl ester (CAS) C21H42O4 358 6.81 0.55
95 2-[5-(2-Hydroxy-propyl)-tetrahydrofuran-2-yl]-propionic acid, t-butyl ester C14H26O4 258 1.7 0.55
96 à-D-Glucopyranoside C20H34O9 418 1.17 0.55
97 1-allyl-2,3-5,6-tetra-o-acetyl-mannofura noside C17H24O9 372 1.12 0.55
98 Mannofuranoside, 1-allyl-2,3-5,6-tetra-O-acetyl C17H24O9 372 1.12 0.55
99 9-Hexadecenoic acid, methyl ester, (Z)- (CAS) C17H32O2 268 37.1 0.79
100 Methyl hexadec-9-enoate C17H32O2 268 26.94 0.79
101 Pentadecanoic acid, 14-methyl-, methyl ester (CAS) C17H34O2 270 13.97 5.54
102 Hexadecanoic acid (CAS) C16H32O2 270 54.59 5.54
103 l-(+)-Ascorbic acid 2,6-dihexadecanoate C38H68O8 652 14.07 5.22
104 9-Octadecenoic acid (Z)- (CAS) C18H34O2 282 4.71 5.22
105 Pentadecanoic acid C15H30O2 242 3.8 5.22
106 Elaidinsaeure methyl ester C19H36O2 296 7.69 14.89
107 Methylelaidate C19H36O2 296 19.42 14.89
108 cis-vaccenic acid C18H34O2 282 16.52 12.28
109 Oleic acid C18H34O2 282 4.9 12.28
110 Heptadecene-(8)-carbonic acid-(1) C18H34O2 282 3.16 12.28
111 Octadecanoic acid, 2-(2-hydroxyethoxy)ethyl ester C22H44O4 372 4.87 0.57
112 Octadecanoic acid (CAS) C18H36O2 284 58.57 0.57
113 Tricosane C23H48 324 11.2 0.83
114 Eicosane C20H42 282 7.46 0.83
115 Pentacosane C25H52 352 7.17 0.83
116 Heneicosane C21H44 296 6.33 0.83
117 Hexatriacontane C36H74 506 6.09 0.83
118 Nonadecane C19H40 268 5.38 0.83
119 Docosane C22H46 310 5.38 0.83
120 Pentatriacontane C35H72 492 5.17 0.83
121 Triacontane C30H62 422 4.36 0.83
122 1-Heptacosanol C27H56O 396 6.62 1.94
123 n-Tetracosanol-1 C24H50O 354 5.6 1.94
124 1-Heneicosanol C21H44O 312 4.94 1.94
125 Z-12-Pentacosene C25H50 350 4.75 1.94
126 9-Hexacosene C26H52 364 3.54 1.94
127 10-Heneicosene (c,t) C21H42 294 3.27 1.94
128 9-Tricosene, (Z)- C23H46 322 3.27 1.94
129 n-Nonadecanol-1 C19H40O 284 2.89 1.94
130 1-Heneicosyl formate C22H44O2 340 2.27 1.94
131 Octacosane (CAS) C28H58 394 5.4 1.38
132 9-Octadecenamide C18H35NO 281 18.5 0.51
133 cis-13-Eicosenoic acid C20H38O2 309 17.07 0.51
134 2-Hexadecanol C16H34O 242 8.48 0.74
135 17-Pentatriacontene C35H70 490 5.81 0.74
136 cis-10-nonadecenoic acid C19H36O2 296 3.73 0.74
137 cis-11-Eicosenoic acid C20H38O2 310 3.44 0.74
138 Erucic acid C22H42O2 338 3.04 0.74
139 1-[(4′á)-3′-Ethylenedioxy-18′-norkaur-15′-en-17′-yl]pyrroli dine C25H39NO2 385 51.45 4.63
140 3á-(Peroxymethyl)-5-vinyl-A,B-bisnor-5á-cholestane C28H48O2 416 15.13 4.63
141 2-Allyl-6-(1,1-dimethylpropyl)-3-n-pentadecylphenol C29H50O 414 9.17 4.63
142 (2S,3S)-2,3-Isopropylidenedioxy-4-tosyloxybutan-1-yl tetrahydropyran ether C19H28O7S 400 6.1 4.63
143 3-O-(trimethylsilyl)-5,7,4′-tri-O-methylkaempferol C21H24O6SI 400 1.4 4.63
144 N,N-Diethyl-1,3-dihydro-1-oxo-3,3-diphenyl-5-isobenzo-f urancarboxamide C25H23NO3 385 0.59 4.63
145 13-Docosenamide C22H43NO 337 60.05 6.91
146 Squalene C30H50 410 10.15 0.63
147 trans-Geranylgeraniol C20H34O 290 4.62 0.63
148 Methyl trisporate C C19H28O4 320 6.92 2.03
149 2-Cyclohexene-1-carboxylic acid C19H28O4 3200 6.92 2.03
150 Thalmiculimine C37H38N2O7 622 3.1 2.03
151 Bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl] maleate C38H56O6 608 2.43 2.03

Fig. 1.

Fig. 1

GC–MS chromatogram of A. bilimbi L. fruit extract performed in the THERMO GC—TRACE ULTRA VER: 5.0, THERMO MS DSQ II machine. Non-polar column DB 5-MS capillary standard, helium gas as a carrier, with an injection volume of 1 μl was used

Table 3.

Major compounds identified by GC–MS in the A. bilimbi L. fruit extract, reported to have biological activity and cited in PUBMED

S. no Compound Activity
1 Hexadecanoic acid, ethyl ester Antioxidant, hypocholesterolemic nematicide, pesticide, anti-androgenic flavour, hemolytic, 5-alpha reductase inhibitor (Kumar et al. 2010)
2 Squalene Chemo-preventive against colon cancer (Rao and Harold 1998)
3 Erucic acid X-linked adrenoleukodystrophy (Rizzo et al. 1989)
4 Oleic acid Reduce blood pressure (Teres et al. 2008)
5 Chimanine D Antileishmanial (Fournet et al. 1993)
6 Boronic acid Potential pharmaceutical agent (selective reduction of aldehydes, enzyme inhibitors, asymmetric synthesis of amino acids) (Yang 2003)
7 5-Hydroxymethyl furfural Against sickle cell anaemia (Lin et al. 2008)
8 Mannitol Used for acute traumatic brain injury (Wakai et al. 2013)
9 Desulphosinigrin Antibacterial (Sabreen et al. 2015)
10 Methyl Pyroglutamate Antibiotic preparation. Smith 1997 in the book Alkaloids: Chemical and Biological Perspectives Chapter 4: Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

Total phenolic content

The total phenolic content of the A. bilimbi L. fruit extract was expressed as gallic acid equivalent in milligram per gram (GAE mg/g) of methanolic fruit extract. The optical density values and its straight line equation (y = mx + c) of standard gallic acid is shown in Fig. 2. The total phenolic content for 250 µg/ml is found to be 209.25 GAE mg/g.

Fig. 2.

Fig. 2

The total phenolic content of A. bilimbi L. fruit extract for 250 µg/ml is 209.25 GAE mg/g

Nitric oxide (NO) scavenging activity

The A. bilimbi L. fruit extract showed an increased nitric oxide scavenging activity with increase of concentration of the extract. Ascorbic acid is used as a standard for determining the IC50 value. Decreased OD values were observed when the concentration of fruit extract increased. The percentage of inhibition is shown in Table 4 and the regression curve for the standard ascorbic acid and A. bilimbi L. extract is shown in Fig. 3, respectively. The IC50 value of A. bilimbi L. fruit extract and standard ascorbic acid was found to be 108.10 and 85.01 which is shown in Table 4.

Table 4.

Percentage inhibition of A. bilimbi L. fruit extract on nitric oxide and its comparison with that of standard ascorbic acid. The IC50 of ascorbic acid is 85.01 and IC50 of A. bilimbi L. extract is 108.10

S. no Concentration (µg) Ascorbic acid % inhibition on nitric oxide A. bilimbi L. fruit extract % inhibition on nitric oxide
1 25 21.10 ± 0.84 2.95 ± 0.88
2 50 33.67 ± 0.87 22.9 ± 1.90
3 75 47.15 ± 1.89 32.70 ± 1.60
4 100 56.74 ± 1.22 46.23 ± 1.56
5 125 63.55 ± 4.67 58.84 ± 1.84
6 150 85.14 ± 1.17 71.09 ± 2.67
IC50 85.01 108.10

Fig. 3.

Fig. 3

A Percentage inhibition of standard (ascorbic acid) at different concentrations on NO B Percentage inhibition of A. bilimbi L. fruit extract at different concentrations on NO

Discussion

Traditionally 6000 plants are used in Indian folk and herbal medication and 3000 plants are in documented medicine used against diseases (Rajshekharan 2002). Their medicinal value is due to the presence of phytochemicals. Phytochemicals are also called as natural products, plant constituents, and secondary metabolites which have medicinal properties to which they belong and the mechanism of action was not known up to the extent. These phytochemicals have great potentialities in drug discovery for various diseases (Justin et al. 2014). The phytochemicals like alkaloid, carbohydrate, glycosides, phenols, flavonoids, saponins, steroids, and tannins compounds are remedy to cure diseases and fight against different kinds of pathogens, as medicine (Hassan et al. 2004). In the current investigation, we have revealed the presence of phytochemicals (alkaloids, carbohydrate, phenols, flavonoids, saponins and tannins) in the A. bilimbi L. fruit extract. Our result on phytochemical presence is consistent with an earlier study (Hasanuzzaman et al. 2013). Moreover, there may be a region-wise difference in the presence of phytochemicals in any plant. Gas chromatographic–mass spectrometry (GC–MS) is a ubiquitous analytical technique of choice in toxicology, environmental research, food science and forensic research. A. bilimbi L. fruit extract was separated by GC and the compounds were identified by the MS by the NIST and Wiley 9.0 libraries. GC–MS analysis revealed the presence of major biologically active compounds (4H-pyran-4-one, 2,3-dihydro3,5-dihydroxy-6-methyl, hexadecanoic acid, squalene, erucic acid, oleic acid, chimanine D, boronic acid, 5-hydroxymethyl furfural, 2-deoxy-d-galactose, mannitol, desulphosinigrin, methyl pyroglutamate) having medicinal important as given in Table 3. We have not identified any steroid compounds in GC–MS report which correlates well with the results of phytochemical screening. Total phenolic compounds present in the A. bilimbi L. fruit extract were determined by Folin–Ciocalteu method. We have also observed a high level of total phenolic compounds in the A. bilimbi L. fruit extract at a concentration of 209.25 GAE mg/g. Presence of phenolic compound apparently explains the antioxidant nature of the plant (Awika et al. 2003) due to its hydroxyl group which have the scavenging activity (Hatano et al. 1989). More and more phenolic compounds are used in foods to improve the nutritional quality (Kahkonen et al. 1999). The presence of benzenoid ring (hydrophobic) and hydrogen bonding in phenolic hydroxyl groups will help in interacting with the proteins, accounting for its potent nature to act as antioxidants (Parr and Bolwel 2002). Free radicals possess high reactive nature; they attack nearest stable molecules like lipids, proteins, DNA and carbohydrates by sneaking their electrons (Patil et al. 2013). Different forms of free radicals are reactive oxygen species (ROS) and reactive nitrogen species (RNS). Antioxidants are the molecules that scavenge free radicals. They safeguard the cell components from the free radicals (Shenoy and Shirwaikar 2002) by scavenging the free radicals by scavenging the ROS and RNS (Rozina et al. 2012). NO is one of the abundant free radicals categorized under RNS. It is a highly reactive nitrogen species formed during inflammations, capable of damaging proteins, lipids and DNA (Valko et al. 2007). Synthetic antioxidants like butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT) and tertiary butyl hydroquinone are used in food supplements. They are used to treat numerous human diseases, but these compounds have toxic effects (Kombo 2000). Plants are the natural sources for the antioxidants they possess high quantity and quality of antioxidants which can scavenge the free radicals (Wang et al. 1996). In the present investigation, the antioxidant activity for A. bilimbi L. fruit extract is proven from its high level of NO scavenging activity similar to the standard ascorbic acid used in our study. The IC50 value of nitric oxide is 85.01, whereas the IC50 value of ascorbic acid is 108.10. Furthermore, boronic acid identified in our GC–MS analysis was reported to have nitric oxide scavenging activity (Yang et al. 2003). This may be one of the reasons for the significant nitric oxide scavenging activity observed in this study for A. bilimbi L. fruit extract.

Conclusion

From the above study, it is apparent that the A. bilimbi L. fruit extract is a rich source of phytochemicals (natural products) with biological activity. The GC–MS report on this fruit proves that natural products have pharmacologically and biologically active compounds. A high phenolic content is observed in our study. A. bilimbi L. fruit extract is also found to have NO scavenging activity in our study.

Acknowledgements

The authors are thankful to the Department of Biosciences and Technology, Karunya University and The South Indian Textile Research Association (SITRA), Coimbatore.

Compliance with ethical standards

Conflict of interest

The author declares that there is no conflict of interest.

Contributor Information

Jagadish Kumar Suluvoy, Email: jagadishbabu77@gmail.com.

V. M. Berlin Grace, Phone: +91-9894051175, Email: berlsdg@gmail.com, Email: berlin@karunya.edu

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