3 Biotech (2017)7:85

DOI 10.1007/s13205-017-0678-9

ORIGINAL ARTICLE

Phytochemical profile and free radical nitric oxide (NO)

scavenging activity of Averrhoa bilimbi L. fruit extract
Jagadish Kumar Suluvoy1 • V. M. Berlin Grace1

Received: 11 January 2017 / Accepted: 27 February 2017

Ó Springer-Verlag Berlin Heidelberg 2017

Abstract Averrhoa bilimbi L. belongs to family Oxali- study. A. bilimbi L. fruit extract is also found to have NO
daceae. Traditionally, people use this plant (root, bark, scavenging activity in our study.
leaves and fruits) for treating several illnesses include
itches, boils, syphilis, whooping cough, hypertension, fever Keywords Averrhoa bilimbi L.  GC–MS  Nitric oxide 
and inflammation. The aim of the study was to evaluate the Phenol  Phytochemical  Antioxidant
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, Introduction
antioxidant scavenging activity and biologically important
compounds were identified by GC–MS analysis. The pre- Everyday 50,000 premature deaths are caused due to
liminary phytochemicals, GC–MS, total phenolic content infectious diseases (Singh et al. 1992; Robin et al. 1998). In
and NO scavenging activity of the plant were analysed. In accordance with the World Health Organization (WHO)
the present investigation, the A. bilimbi L. fruit extract has 2014 diseases like malaria, dengue, leishmaniasis, Lyme
major phytochemicals. Among the 151 compounds iden- disease, tuberculosis, schistosomiasis, and yellow fever,
tified in GC–MS, 15 compounds are found to have diverse carried by mosquitoes, flies, ticks, water snails and air
biological activity. We also observed that the A. bilimbi L. infect one billion people and more than one million people
fruit extract has high level of total phenolic compounds at a will die. Pathogens and diseases become drug resistant and
concentration of 209.25 GAE mg/g. Presence of phenolic the best alternate approach are plants to eliminate diseases
compound apparently explains the antioxidant activity of and therapeutic complications (Fabricant and Farnsworth
the plant. Antioxidant activity of A. bilimbi L. fruit extract 2001). From time immemorial plants are used as medicine
is proven from its high level of NO scavenging activity of to treat diseases. Before the discovery of allopathy humans
potent IC50 value of 108.10. From the above study, it is depended on Ayurveda and homeopathy medicine which
apparent that the A. bilimbi L. fruit extract is a rich source are completely based on plants and herbs. These herbs and
of phytochemicals (natural products) with biological plant materials act as medicine to cure diseases (Nostro
activity. The GC–MS report on this fruit proves that natural et al. 2000). Tribal people depend on the rich diversity of
products have pharmacologically and biologically active forest to overcome the health care needs. Forests have
compounds. A high phenolic content is observed in our excellent vegetation (flora) with high quality of medicinal
value (Kadhirvel et al. 2010). Phytochemicals are the non-
nutrient compounds with beneficial health effects leading
& V. M. Berlin Grace to pharmacological importance and are used in medication
[email protected]; [email protected]
(Nisa et al. 2011). Fruits play major role in human diet due
Jagadish Kumar Suluvoy to their bioactive compounds, natural sugars and organic
[email protected]
acids with relatively high antioxidant activity (Rechkem-
1
Department of Biotechnology, Karunya University, Karunya mer 2001) and are a rich source of vitamins (A, B6, C, E,
Nagar, Coimbatore, Tamil Nadu, India niacin, and thiamine) dietary fibre and minerals

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(Wargovich 2000). Averrhoa bilimbi L. is a long-lived proteins and also deaminate DNA bases (Umamaheswari
green plant which gives edible fruits, belonging to the and Chatterjee 2008). Hence it is essential to reduce the
family Oxalidaceae–Oxalis and grows 16–33ft (5–10 m) in levels of NO in human body. Besides to the reactive oxy-
height, with short trunk dividing into number of upright gen species (ROS) NO was found to be elevated in
branches. It is found throughout Malaysia, Indonesia, inflammation (Baygutalp et al. 2015), colon cancer (Erd-
Myanmar, Bangladesh, Srilanka and common in Southeast man et al. 2009) and pathological conditions like gas-
Asian countries (Rahman et al. 2014). In India it is avail- trointestinal disorders (Cho 2001). The traditional usage of
able mostly in Kerala regions, particularly the Kani tribal the fruits of A. bilimbi L. for anti-inflammation, anti-dia-
traditional healers in Thodu hills region (Kerala) use the betic and anti-hypertensive was highlighted in a report on
raw leaves and fruits of A. bilimbi L. plant for ailments in Malaysian medicinal plants (Harun et al. 2015). A. bilimbi
circulatory system (Xavier et al. 2014) and the local name L. is a rich source of vitamin C, A, B1 and 100 g of edible
is Irumban puli or Pulingi. The parts like bark, leaves, portion was found to have moisture, 94.2–94.7 g; fibre,
seeds, flowers, fruits, roots and the entire A. bilimbi L. plant 0.6 g; ash, 0.31–0.40; protein, 0.61 g; calcium, 3.4 g; iron,
is used as alternative medicine to treat numerous diseases 1.01 mg; riboflavin, 0.32 mg; thiamine, 0.010 mg; ascorbic
majorly as anti-diabetic agent (Kumar et al. 2013). Tradi- acid, 15.5 mg (Zakaria et al. 2007). Ascorbic acid has been
tionally it is used in medication to cure cough, cold, boils, used as standard drug for the estimation of nitric oxide
itches, syphilis, whooping cough, rheumatism and hyper- scavenging activity (singh et al. 2012). As the A. bilimbi L.
tension (Sabiha et al. 2012). A. bilimbi L. shows antimi- fruit extract has also shown a good antioxidant potential
crobial activity against gram positive and gram negative against DPPH (Chauhan and Kapfo 2013), we made an
bacteria (Karon et al. 2011), antifungal activity (Nazmul attempt to analyse its in vitro NO scavenging activity.
et al. 2011), cytotoxic activity (Das et al. 2011), anti-dia-
betic activity (Pushparaj et al. 2000) and the leaves of A.
bilimbi L. could increase the serum insulin level (Patel Materials and methods
et al. 2012) in diabetes mellitus. Administration of A. bil-
imbi L. fruit (toxicity studies) extract 1 g/kg bw did not Chemicals and reagents
affect the mice (Savithri et al. 2009). However, in spite of
having of such a great traditional medicinal use, the All the chemicals used for this experiment were analytical
knowledge on its phytochemical is limited. Few studies of grade purchased from Hi-Media, and SD Fine Chemicals.
phytochemicals on the A. bilimbi L. fruit extract have
shown contradictory data on the presence of alkaloids, Selection and authentication of fruit
tannins, glycosides, saponins and steroids (Sabiha et al.
2012). This study is therefore designed to analyse the Averrhoa bilimbi L. fruit samples were collected from
active phytochemicals present in the fruits of A. bilimbi L. Palakkad district, Kerala, during February to March (2015).
by biochemical tests and GC–MS which may be useful for The fruits of A. bilimbi L. are used as a source of food and
exploring its ethno-pharmacological significance and to medicine by tribes and settler communities of the local
validate scientifically its medicinal properties. Several people. The authentication of the fruit was done by the
previous studies have shown the free radical scavenging Botanical Survey of India (BSI) Coimbatore, Tamilnadu,
activity of A. bilimbi L. fruit extract through DPPH scav- India. The authentication number given by the BSI is BSI/
enging activity (Asna and Noriham 2014; Sabiha et al. SRC/5/23/2015/Tech.
2012). Oxidative stress has major action in human anat-
omy, physiology and diseases like cardiovascular diseases, Extraction of fruit material
diabetes, inflammatory conditions, ageing and cancer
(Joyce 1987). Nitric oxide (NO) plays a major role in The fruits of the A. bilimbi L. were collected, air dried
several in vivo diseases like neuronal signalling, smooth and made into fine powder by the mortar and pestle.
muscle relaxation, regulation of cell-mediated toxicity and Extraction from the fruits was done according to the
inhibition of platelet aggregation (Hagerman et al. 1998). method described by Singh et al. (2012). The powder
Surplus NO is reported to direct DNA fragmentation, cell (25 g) was used for the extraction with 250 ml of
damage and neuronal cell death. NO will not affect the methanol (95% v/v) in a soxhlet apparatus. The remaining
DNA and proteins directly but NO is very unstable in methanol was evaporated using rotary evaporator. The
aerobic condition and produces NO2, N3O4, N2O4 inter- obtained thick semi-solid crude extract was stored at
mediates which are genotoxic, affecting the DNA repair 2–4 °C for further use.

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Phytochemical screening of A. bilimbi L. fruits Test for tannins

The A. bilimbi L. fruit (methanol) extract was analysed for 0.1gm of crude extract of A. bilimbi L. fruit was mixed in
the presence of alkaloid, carbohydrate, glycosides, phenols, distilled water and filtered. Few drops of ferric chloride
flavonoids, saponins, steroids and tannins using the solution were added to the filtrate. The green or blue-green
respective biochemical tests as follows. precipitate indicated the presence of tannins (Trease and
Evans 2002).
Test for alkaloids

Two millilitre of 1% HCl was mixed with 0.1 gm of crude GC–MS analysis on A. bilimbi L. fruit extract
extract and heated slightly. After cooling Wagner’s reagent
and Mayer’s reagent were added to it. The presence of Averrhoa bilimbi L. methanolic fruit extract was sub-
buff-coloured precipitate indicated the presence of alka- jected to gas chromatography–mass spectroscopy (GC–
loids (Sofowora 1993). 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
Test for carbohydrates
of 70 eV were used. The temperature of the column was
set to 80–250 °C at 8 °C/min rate. Temperature of 280
Benedict’s reagents was mixed with the 0.1 gm of crude
and 290 °C were set for the GC injector and MS transfer,
extract and slightly boiled, appearance of reddish brown
respectively. Helium was used as a carrier gas at a flow
precipitate indicated the presence of the carbohydrates
rate of 1.0 ml/min. The sample volume of 1 ll was used
(Harborne 1973).
for analysis. By the retention time and mass fragmenta-
tion patterns, the major compounds present in the fruit
Test for flavonoids extract were analysed. The National Institute of Standards
and Technology (NIST) and Wiley 9.0 library was used
The appearance of pink scarlet colour when 0.1 gm of (Sakthivel and Guruvayoorappan 2013) for the detection
crude extract was mixed with few drops of concentrated of compounds.
HCl and Mg pellets indicated the presence of flavonoids
(Odebiyi and Sofowora 1978).
Estimation of phenols in A. bilimbi L. fruit extract
Test for phenols
The total phenolic compounds were estimated using the
Two millilitre of 2% ferric chloride was mixed with the Folin–Ciocalteu reagent (Slinkard and Singleton 1977).
0.1 gm of crude extract and the presence of blue-green or Briefly 0.1 ml of A. bilimbi L. fruit extract of different
black coloration indicated the presence of phenols (Yadav concentrations (50, 100, 150, 200, and 250 lg/ml) were
and Agarwala 2011). mixed with 2 ml of 10% Folin–Ciocalteu reagent and
3 ml of 7% Na2 CO3 was added. This was incubated for
Test for saponins 30 min at room temperature and the absorbance was
measured using UV-spectrophotometer at 760 nm.
Saponin presence was detected by the frothing test. Briefly Gallic acid was used as standard and all the results were
0.1 gm of crude extract was mixed well in water and performed in triplicates. The total phenol concentration
shaken, the appearance of foam indicated the preliminary is expressed in mg gallic acid equivalent (GAE).
evidence for the presence of saponins (Kumar et al. 2009).

Nitric oxide (NO) scavenging activity of A. bilimbi

Test for steroid (Liebermann test) L. fruit extract
0.1 gm of crude extract was mixed with 2 ml H2SO4 and The nitric oxide (NO) scavenging activity of the A. bilimbi
slowly added to 2 ml of acetic anhydride. The colour L. fruit extract was expressed in percentage inhibition
change from violet to green or blue indicated the presence (Vaijanathappa et al. 2008). Briefly 3 ml of 10 mM sodium
of steroids (Edeoga et al. 2005). nitroprusside (0.5 mM PBS pH 7.4) was mixed with 1 ml

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of A. bilimbi L. fruit extract at different concentrations (25, analysis reported the presence of various phenol, flavonoid,
50, 75, 100, 125, and 150 lg/ml) and incubated at 25 °C lipid, alkaloid and acid compounds which were shown in
for 150 min. Then 0.5 ml of the reaction mixture was basic phytochemical screening test.
removed and 1 ml of sulfanilic acid reagent (0.33% in 20%
glacial acetic acid) was added and again incubated for Total phenolic content
5 min at 25 °C. After adding 1 ml of naphthyl ethylene
diamine dichloride (0.1 w/v), the entire reaction mixture The total phenolic content of the A. bilimbi L. fruit extract
was allowed to stand for 30 min at room temperature. The was expressed as gallic acid equivalent in milligram per
absorbance was measured at 540 nm. Similar procedure gram (GAE mg/g) of methanolic fruit extract. The optical
was repeated for the standard ascorbic acid at different density values and its straight line equation (y = mx ? c)
concentrations (25, 50, 75, 100, 125, 150 lg/ml). The same of standard gallic acid is shown in Fig. 2. The total phe-
reaction mixture with the methanol served as control nolic content for 250 lg/ml is found to be 209.25 GAE
(without extract and standard) mg/g.
%Inhibition ¼ ðA0  A1 Þ=A0  100;
Nitric oxide (NO) scavenging activity
where A0 is the absorbance of the control and A1 is the
absorbance of the sample. 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 deter-
Results mining the IC50 value. Decreased OD values were observed
when the concentration of fruit extract increased. The
Phytochemical analysis percentage of inhibition is shown in Table 4 and the
regression curve for the standard ascorbic acid and A.
Preliminary phytochemical tests revealed the presence of bilimbi L. extract is shown in Fig. 3, respectively. The
alkaloids, carbohydrates, phenols, flavonoids, saponins and IC50 value of A. bilimbi L. fruit extract and standard
tannins (Table 1). The presence of more phenols was ascorbic acid was found to be 108.10 and 85.01 which is
observed in the preliminary screening. The test for sterols shown in Table 4.
answered negative in our study.
Discussion
Phytochemical compounds identified by GC–MS
analysis Traditionally 6000 plants are used in Indian folk and herbal
medication and 3000 plants are in documented medicine
By comparing with the National Institute of Standards and used against diseases (Rajshekharan 2002). Their medici-
Technology (NIST) and Wiley 9.0 library, the major nal value is due to the presence of phytochemicals. Phy-
compounds are identified and listed in Table 2. The GC– tochemicals are also called as natural products, plant
MS chromatogram is shown in Fig. 1. Among the 151 constituents, and secondary metabolites which have
compounds identified, 15 compounds are found to have medicinal properties to which they belong and the mech-
various biological activity which were reported from other anism of action was not known up to the extent. These
studies as mentioned in Table 3. Furthermore, the GC–MS phytochemicals have great potentialities in drug discovery
for various diseases (Justin et al. 2014). The phytochemi-
Table 1 The phytochemicals present in A. bilimbi L. fruit extract. It cals like alkaloid, carbohydrate, glycosides, phenols, fla-
reveals the presence of alkaloids, carbohydrates, phenols, flavonoids, vonoids, saponins, steroids, and tannins compounds are
saponins, tannins. (?? indicates more amount) and the absence of
remedy to cure diseases and fight against different kinds of
steroids (-)
pathogens, as medicine (Hassan et al. 2004). In the current
S. no Test Results investigation, we have revealed the presence of phyto-
1 Alkaloids ? chemicals (alkaloids, carbohydrate, phenols, flavonoids,
2 Carbohydrates ? saponins and tannins) in the A. bilimbi L. fruit extract. Our
3 Phenols ?? result on phytochemical presence is consistent with an
4 Flavonoids ? earlier study (Hasanuzzaman et al. 2013). Moreover, there
5 Saponins ?
may be a region-wise difference in the presence of phy-
6 Steroids -
tochemicals in any plant. Gas chromatographic–mass
spectrometry (GC–MS) is a ubiquitous analytical technique
7 Tannins ?
of choice in toxicology, environmental research, food

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Table 2 Compounds identified by GC–MS in the A. bilimbi L. fruit extract

S. Compound Empirical Empirical Probability Area
no formula weight %

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

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Table 2 continued
S. Compound Empirical Empirical Probability Area
no formula weight %

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- C13H17Cl5N4 404 2.26 0.83
trichloromethylpyrimidine
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 40 -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

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Table 2 continued
S. Compound Empirical Empirical Probability Area
no formula weight %

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-[(40 á)-30 -Ethylenedioxy-180 -norkaur-150 -en-170 -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)-5,7,40 -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

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85 Page 8 of 11 3 Biotech (2017)7:85

Table 2 continued
S. Compound Empirical Empirical Probability Area
no formula weight %

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 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 ll was
used

science and forensic research. A. bilimbi L. fruit extract level of total phenolic compounds in the A. bilimbi L. fruit
was separated by GC and the compounds were identified extract at a concentration of 209.25 GAE mg/g. Presence of
by the MS by the NIST and Wiley 9.0 libraries. GC–MS phenolic compound apparently explains the antioxidant
analysis revealed the presence of major biologically active nature of the plant (Awika et al. 2003) due to its hydroxyl
compounds (4H-pyran-4-one, 2,3-dihydro3,5-dihydroxy-6- group which have the scavenging activity (Hatano et al.
methyl, hexadecanoic acid, squalene, erucic acid, oleic 1989). More and more phenolic compounds are used in
acid, chimanine D, boronic acid, 5-hydroxymethyl furfural, foods to improve the nutritional quality (Kahkonen et al.
2-deoxy-D-galactose, mannitol, desulphosinigrin, methyl 1999). The presence of benzenoid ring (hydrophobic) and
pyroglutamate) having medicinal important as given in hydrogen bonding in phenolic hydroxyl groups will help in
Table 3. We have not identified any steroid compounds in interacting with the proteins, accounting for its potent
GC–MS report which correlates well with the results of nature to act as antioxidants (Parr and Bolwel 2002). Free
phytochemical screening. Total phenolic compounds pre- radicals possess high reactive nature; they attack nearest
sent in the A. bilimbi L. fruit extract were determined by stable molecules like lipids, proteins, DNA and carbohy-
Folin–Ciocalteu method. We have also observed a high drates by sneaking their electrons (Patil et al. 2013).

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3 Biotech (2017)7:85 Page 9 of 11 85

Table 3 Major compounds S. no Compound Activity

identified by GC–MS in the A.
bilimbi L. fruit extract, reported 1 Hexadecanoic acid, ethyl Antioxidant, hypocholesterolemic nematicide, pesticide, anti-
to have biological activity and ester androgenic flavour, hemolytic, 5-alpha reductase inhibitor
cited in PUBMED (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

one of the abundant free radicals categorized under RNS. It

Gallic acid is a highly reactive nitrogen species formed during
0.5 inflammations, capable of damaging proteins, lipids and
0.45
0.4 DNA (Valko et al. 2007). Synthetic antioxidants like
0.35 butylated hydroxyl anisole (BHA), butylated hydroxyl
0.3
OD 0.25 toluene (BHT) and tertiary butyl hydroquinone are used in
Values y = 0.0012x + 0.1549
0.2 R² = 0.9929 food supplements. They are used to treat numerous human
0.15
0.1 diseases, but these compounds have toxic effects (Kombo
0.05 2000). Plants are the natural sources for the antioxidants
0
they possess high quantity and quality of antioxidants
0 50 100 150 200 250 300
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
Fig. 2 The total phenolic content of A. bilimbi L. fruit extract for
scavenging activity similar to the standard ascorbic acid
250 lg/ml is 209.25 GAE mg/g
used in our study. The IC50 value of nitric oxide is 85.01,
Different forms of free radicals are reactive oxygen species whereas the IC50 value of ascorbic acid is 108.10. Fur-
(ROS) and reactive nitrogen species (RNS). Antioxidants thermore, boronic acid identified in our GC–MS analysis
are the molecules that scavenge free radicals. They safe- was reported to have nitric oxide scavenging activity (Yang
guard the cell components from the free radicals (Shenoy et al. 2003). This may be one of the reasons for the sig-
and Shirwaikar 2002) by scavenging the free radicals by nificant nitric oxide scavenging activity observed in this
scavenging the ROS and RNS (Rozina et al. 2012). NO is study for A. bilimbi L. fruit extract.

Table 4 Percentage inhibition S. no Concentration (lg) Ascorbic acid % inhibition A. bilimbi L. fruit extract %
of A. bilimbi L. fruit extract on on nitric oxide inhibition on nitric oxide
nitric oxide and its comparison
with that of standard ascorbic 1 25 21.10 ± 0.84 2.95 ± 0.88
acid. The IC50 of ascorbic acid
2 50 33.67 ± 0.87 22.9 ± 1.90
is 85.01 and IC50 of A. bilimbi
L. extract is 108.10 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

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85 Page 10 of 11 3 Biotech (2017)7:85

Fig. 3 A Percentage inhibition A B

of standard (ascorbic acid) at 100 Ascorbic acid A. bilimbi fruit
different concentrations on NO 80
80
B Percentage inhibition of A. 60
60
bilimbi L. fruit extract at % Inhibition y = 0.4793x + 9.2896 40
different concentrations on NO on NO 40 R² = 0.9798
20 20 y = 0.5281x - 7.082
R² = 0.9921
0
0
0 50 100 150 200
0 50 100 150 200
Concentration in micro gram

Conclusion Erdmana SE, Rao VP, Poutahidisa T, Rogersa AB, Taylora CL,
Jackson EA, Ge Z, Lee CW, Schauera DB, Wogan GN,
Tannenbaumc SR, Foxa JG (2009) Nitric oxide and TNF-a
From the above study, it is apparent that the A. bilimbi L. trigger colonic inflammation and carcinogenesis in Helicobacter
fruit extract is a rich source of phytochemicals (natural hepaticus-infected, Rag2-deficient mice. PNAS
products) with biological activity. The GC–MS report on 106(4):1027–1032
Fabricant DS, Farnsworth NR (2001) The value of plants used in
this fruit proves that natural products have pharmacologi-
traditional medicine for drug discovery. Environ Health Perspect
cally and biologically active compounds. A high phenolic 1(109):69–75
content is observed in our study. A. bilimbi L. fruit extract Firenzuoli F, Gori L (2001) Herbal medicine today: clinical and
is also found to have NO scavenging activity in our study. research issues. eCAM 4(S1):37–40
Fournet A, Barrios AA, Victoria M, Reynald H, Andre C, Jean B
(1993) 2-Substituted quinoline alkaloids as potential antileish-
Acknowledgements The authors are thankful to the Department of
manial drugs. Antimicrob Agents Chemother 37:859–863
Biosciences and Technology, Karunya University and The South
Hagerman AE, RiedK M, Jones GA, Sovik KN, Ritchard NT,
Indian Textile Research Association (SITRA), Coimbatore.
Hartzfeld PW (1998) High molecular weight plant polyphenolics
(tannins) as biological antioxidants. J. Agric. And Food Chem.
Compliance with ethical standards
46:1887–1892
Harborne JB (1973) Phytochemical methods: a guide to modern
Conflict of interest The author declares that there is no conflict of
technique of plant analysis. Chapman & Hall, London
interest.
Harun NH, Septama AW, Jantan I (2015) Immunomodulatory effects
of selected Malaysian plants on the CD18/11a expression and
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