© February 2016 | IJIRT | Volume 2 Issue 9 | ISSN: 2349-6002

ANTI NUTRIENT AND PHYTOCHEMICAL

SCREENING OF AN UNDERUTILIZED FRUIT
SEED: LIMONIA ACIDISSIMA

L. Banupriya, T. Poongodi Vijayakumar

School of Professional Studies, Department of Food Science and Nutrition, Periyar University,
Salem, Tamil Nadu, India.

Abstarct- The phytochemical analysis of the medicinal definite physiological action on human body
plants are important and have commercial interest in (Edeoga et al., 2005). These chemical substances
both research institutes and pharmaceutical are called phytochemicals (Sood et al., 2012). The
companies for the manufacturing of the new drugs
word phyto is a Greek word phyto meaning plant.
for treatment of various diseases. Wood apple fruit
Their function is to provide odor to the plant
has got high medicinal value. The present study was
aimed to investigate the anti-nutrient and preliminary (terpenoids), pigmentation (tannins and quinines)
phytochemical screening of the wood apple seeds, an and flavor (capsacin) (Mallikharjuna et al., 2007).
unconventional protein source. Three different They also help in natural defense mechanism of
extracts (methanol, aqueous and ethanol) were plants. These bioactive compounds are responsible
prepared and analysed for phytochemical for antimicrobial activity of plant extracts in vitro.
constituents. The results revealed the presence of Some common examples of phytochemicals are
medicinally important phytochemical constituents flavonoids, alkaloids, saponins, glycosides,
were high in aqueous extracts of Limonia acidissima
terpenoids, tannins, sterols and carbohydrates
seeds than in methanol and ethanol extractions. The
(Sood et al., 2012).
anti-nutrients (Tannin, Saponin, Oxalate and
Phytate) compositions were higher in wood apple seed Recent epidemiological and controlled-
flour when compared to wood apple seed protein case studies reported that many anti-nutrients that
concentrate. Thus, the results scientifically validate present in a low level give beneficial effects for
the use of Limonia acidissima in the traditional prevention of diseases like coronary diseases and
medicine and it can be used to treat various disorders cancers (Pandey and Rizvi, 2010). Due to this, they
caused by free radical and chemical substances due to can be considered as anti-nutritional factors with
presence of secondary metabolites. negative effects or non-nutritive compounds with
Index Terms- Phytochemical, anti nutrients, wood positive effects on health (Habtamu and Negusse,
apple seed flour (WSF), wood apple seed protein 2014). Natural polyphenols exert their beneficial
concentrate (WSPC), aqueous extract health effects by their antioxidant activity, these
I. INTRODUCTION compounds are capable of removing free radicals,
chelate metal catalysts, activate antioxidant
The interest in natural antioxidants has enzymes, reduce α-tocopherol radicals, and inhibit
increased considerably in recent years because oxidases (Amic et al., 2003; Alia et al., 2003)
of their beneficial effects of prevention and risk Limonia acidissima (syn. Feronia
reduction in several diseases. Phenolic elephantum, Feronia limonia, Hesperethusa
compounds are biologically active substances, crenulata, Schinus limonia) is the only species of
which are considered to be natural antioxidants its genus, in the family Rutaceae. It is native in
(Rybarczyk and Amarowicz, 2007, Duenas et al., Bangladesh, India, Pakistan, Sri Lanka, and
2009).The preliminary screening tests may be Vietnam. Vernacular names in English include:
useful in the detection of the bioactive principles wood-apple, elephant-apple, monkey fruit, and curd
and subsequently may lead to the drug discovery fruit are the variety of common names in the
and development (Kavitha et al., 2013). The languages of its native habitat regions (Nguyen Thi
medicinal value of plants is attributed to the and Nguyen Phuoc, 2014). Limonia acidissima is a
presence of some chemical substances (bioactive large tree growing to 9 metres (30 ft) tall, with
non-nutrient plant compounds) which produce a

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rough, spiny bark. The leaves are pinnate, with 5-7 stirred for 2 hrs at room temperature, and then the
leaflets, each leaflet 25–35 mm long and 10–20 mixture was left overnight. In the next day, the
mm broad, with a citrus-scent when crushed. The mixture was filtered and the residue was collected
fruit is a berry 5–9 cm diameter, and may be sweet and dried for 12 hrs at room temperature. The
or sour. It has a very hard rind which can be process was repeated two more times to extract the
difficult to crack open, and contains sticky brown complete oil from the sample. After sieving (60
pulp and small white seeds (Prasanta and Debasis, mesh), the sample (WSPC) was kept in the
2014). refrigerator for further analysis.
Wood apple has got high medicinal value.
Every part of the fruit posse’s medicinal property. Anti-nutrients determination
Wood apple has anti-diabetic and antioxidant Tannin
potential by reducing the level of blood glucose and Tannin was determined using the method
malondialdehyde (Patel et al., 2012). Wood apple of Trease and Evans, (1978). 1 ml of the
fruit is considered to be one of the natural sources methanolic extract was treated with 5 ml Folin
of anti-oxidants due to its potential radical Dennis reagent in a basic medium and allowed to
scavenging activity of various phytochemicals stand for colour development. The absorbance of
(Nithya and Saraswathi, 2010) and its anti-oxidant the reaction mixture of each sample was measured
properties using different extracts were extensively at 760 nm spectrophotometrically.
studied (Sachin and Arya, 2013, Ramdas and Oxalate
Seema, 2010, Suree Nanasombat et al., 2012 and Total oxalate was determined according to
Teeica Priya Darsini et al., 2013). The Day and Underwood, (1986) procedure. To 1 g of
phytochemical analysis and antibacterial acivity of the ground powder, 75 ml of 15 N H2SO4 was
Limonia acidissima L. plant parts were studied added. The solution was carefully stirred
(Asha Thomas and Ponnammal, 2005). The seed intermittently with a magnetic stirrer for 1 h and
composition and fatty acid profile were reported as filtered using Whatman No 1 filter paper. 25 ml of
28% protein and 34% oil (Ramakrishna et al., the filtrate was then collected and titrated against
1979). But there is little or no information on the 0.1 N KMnO4 solutions till a faint pink colour
phytochemical properties and anti-nutrients of this appeared that persisted for 30 second.
nutritious seed. Hence, the present work evaluates Phytate
the levels of some anti-nutritional factors and Phytate was determined using Reddy and
qualitative phytochemical analysis of poorly Love, (1999) method. 4 g of the ground sample was
utilized wood apple seed. soaked in 100 ml of 2% HCl for 5 h and filtered.
To 25 ml of the filtered, 5 ml 0.3% ammonium
II. MATERIAL AND METHODS
thiocyanate solution was added. The mixture was
Preparation of Wood Apple Seed Flour (WSF) then titrated with Iron (III) chloride solution until a
and Wood Apple Seed Protein Concentrate brownish-yellow color that persisted for 5 min was
(WSPC) obtained.
Ripe wood apple (Limonia acidissima) Saponin
with hard shell, fairly large and globular shaped Saponin was determined using the method
with soft, fleshy, brownish edible pulp was selected of Birk et al., (1963) as modified by Hudson and
for the study. They were purchased from the local El-Difrawi, (1979). 20 ml of 20% aqueous ethanol
market of Salem as a bulk for analysis. The fruits was added to 10 g of the ground sample and
were botanically identified by the botanist at the agitated with a magnetic stirrer for 12 h at 55°C.
Taxonomy section, Department of Environmental The solution was then filtered using Whatman No.1
Sciences, Periyar University, Salem, Tamilnadu. filter paper and the residue reextracted with 200 ml
From the fruit, the seeds were manually separated 20% aqueous ethanol. The extract was reduced to
by washing under running water. The wood apple 40 ml under vacuum and 20 ml diethyl ether added
seeds were sun dried, grinded into flour (WSF) and in a separating funnel and shaken vigorously. The
packed in tight polythene bags for further analysis aqueous layer was recovered and ether layer
at room temperature. The other part of seeds was discarded. The pH of the aqueous solution was
defatted using El-Tinay et al., (1988) method. adjusted to 4.5 by adding NaOH, and the solution
Sample was mixed with n-hexane (1:10 w/v) and shaken with 60 ml n-butanol. The combined

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 © February 2016 | IJIRT | Volume 2 Issue 9 | ISSN: 2349-6002

butanol extracts were washed twice with 10 ml of of analysis (Olayinka and Anthony, 2011; Kaur and
5% aqueous NaCl and evaporated to dryness in a Arora, 2011; Sharma et al., 2011). The reactions in
fume cupboard to give a crude saponin which was this analysis revealed the presence or absence of
weighed. these compounds in the seed with different
Preparation of the seed extracts extractions.
Preparation of WSF and WSPC extracts
III. RESULTS AND DISCUSSION
using different solvents (methanol, aqueous and
ethanol) was done according to a combination of Anti nutrient composition
the methods used by Pizzale et al., (2002) and Lu The results of anti-nutritional composition
and Foo, (2001). of wood apple seed flour and protein concentrate
Qualitative phytochemical analysis samples are shown in Table 1. The tannin content
Phytochemicals like flavonoids and in wood apple seed flour was higher than WSPC
phenols are strong antioxidants and have an (0.89 and 0.65 mg/100g) respectively. The results
important role in the health care system (Dhan when compared to soaked tamarind seed (STS),
Prakash et al., 2007). Screening of active STS had higher tannin content with 4.84% (Linda
compounds from plants has led to the discovery of et al., 2014). The lower content of tannin in sample
new medicinal drugs which have efficient STS can be attributed to the fact that tannin are
protection and treatment roles against various water soluble compounds which makes them easy
diseases, including cancer and alzheimer’s disease to be eliminated by soaking (Siddhuraju et al.,
(Soma et al., 2010). Qualitative phytochemical 1995). Tannins are astringent in taste and help in
screening was done for evaluation of major healing of wounds and inflamed mucous membrane
phytochemical constituents such as tannins, (Njoku and Akumefula, 2007). Tannins are
saponins, flavonoids, sterols, terpenoids, potential metal ion chelator, proton precipitating
carbohydrates, phenols, proteins and amino acids, agents and biological antioxidant (Okonkwo,
alkaloids and glycosides using standard procedure 2009).

Table 1: Anti- nutrient composition of wood apple seed flour and wood apple seed protein concentrate
(mg/100g)
Anti-nutrients WSF WSPC
Tannin 0.89+0.01 0.65+0.02
Phytate 0.60+0.00 0.38+0.00
Oxalate 0.33+0.05 0.03+0.00
Saponin 0.45+0.00 0.21+0.00
Data are mean values ± standard deviation (SD) of duplicate results

The phytate content was also higher in benefits such as antioxidant, anticancer,
wood apple seed flour (0.60 mg) than in wood hypocholesterolemic and hypolipidemic effects. In
apple seed protein concentrate (0.38 mg). These fruits and vegetables, phytic acid helps to prevent
amounts are very low when compared with those oxidative browning by inhibiting polyphenol
reported for some commonly used legumes, oxidase. It may be used as a safe preservative and
cowpea (2-2.9%), pigeon pea (2-2.4%) and African antioxidant in food products by suppressing
yam beans (2.4%) (Oboh, 2006). Phytic acid binds oxidative reactions catalyzed by iron (Fasidi and
calcium, iron, zinc and other minerals, thereby Olorunmaiye, 1994). The decrease in phytic acid
reducing their availability in the body (FAO, 1990). content by soaking, cooking of pre-soaked bean or
It also inhibits protein digestion by forming germination may be due to leaching out of this
complexes with them. However, the phytate compound in water (Osman, 2007).
content can further be lowered by processing Oxalate level was comparatively low in
(Esenwah and Ikenebomeh, 2008) and has been wood apple seed flour (0.33mg/100g) as (3.06%)
considered as an anti-nutritional component in present in M. scandenis, and it is known to cause
cereals, seeds and beans. However, resent research great risk of renal absorption. Heat treatment
have shown that phytic acid has many health (cooking) has been found to be an effective

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measure in reducing the oxalate levels in leafy difference in the reduction of the anti-nutrients
vegetable, thus, making the food prepared from concentrations and toxicants present in Mucuna
these accessions safe for human consumption pruriens (Velvet Beans) seeds (Nwaoguikpe et al.,
(Lumu and Katongole, 2011). 2011)
The saponin in wood apple seed flour and Preliminary phytochemical screening
wood apple seed protein concentrate was 0.45 mg Phytochemical constituents such as
and 0.21 mg/100g respectively. According to tannins, flavonoids, alkaloids and several other
Harborne (1984), saponins have anti-hyper aromatic compounds or secondary metabolites of
cholesterol, anti- inflammatory, anti-mutagenic, plants serve as defense mechanism and curative
cardiac depressant property and also appear to kill properties against predation by many
or inhibit cancer cells without killing the normal microorganism, insects and herbivores (Britto and
cells in the process (Okwu and Josiah, 2006; Nafiu Sebastiana, 2011). The analysis of phytochemical
et al., 2011), while some others have reported the screening of wood apple seed flour and wood apple
expectorant action (Ayoola and Adeyeye, 2010). seed protein concentrate was showed in Table 2.
Plants containing saponins are used to heal wounds The different extractions (methanol, aqueous and
(Okwu and Josiah, 2006) because saponins have ethanol) revealed the presence and absence of
the ability to precipitate and coagulate red blood phyto constituents of the wood apple seed. The
cells (Sood et al., 2012). The present study results major compounds were present in aqueous
were lower than Cassipourea congoensis seeds, extractions of WSF and WSPC rather than
which were rich in oxalate (10.21+1.11), tannin methanol and ethanol extractions. The protein,
(2.84+0.12) and saponin (7.17+0.18) (Nkafamiya et carbohydrate, amino acid and fats were highly
al., 2007) and leguminous seeds (Mucuna Ghana, present in wood apple seed flour when compared to
Mucuna preta and Mucuna Veracruz mottle) had wood apple seed protein concentrate. This result
higher content of oxalate (8.31+0.03), phytic acid was may be due to defatting of wood apple seed
(85.47+0.62) and tannin (10.30+1.15) (Amoo et al., flour. Phenolic compounds could be a major
2009). determinant of antioxidant potentials of food plants
Some of these anti-nutrients can be and have been associated with the health benefits
reduced by processing and cooking, for example, derived from consuming high levels of fruits and
boiling can reduce the soluble oxalate content of a vegetables. Similar results were found in Solanum
food, if the water used for boiling is discarded indicum Linn., which has high preservation
(Odugbemi, 2006). The toxic and anti-nutrient capacity and nutritional values, because total
effects of compounds in the plants could be phenolic compounds prevent from damage of
removed by several processing methods such as nutrients contain double bonds such fatty acids,
soaking, germination, boiling, autoclaving, flavor compounds even proteins and amino acids
fermentation, genetic manipulation and other and other compounds (Kahkonen et al., 1999). But
processing methods (Soetan, 2008; Enechi and maximum phenolic content was found in
Odonwodu, 2003). The effect of processing such methanolic extract than in aqueous extract in Aegle
as soaking, soaking and boiling, had significant marmelos seeds (Ganesh et al., 2011).

Table 2: Phytochemical screening of wood apple seed flour and wood apple seed protein concentrate
WSF WSPC
Ethanol Aqueous Methanol Ethanol Aqueous Methanol
Phytochemicals
extract extract extract extract extract extract
Alkaloids + ++ ++ + + ++
Flavonoids ++ +++ + ++ +++ +
Amino acids + +++ + + +++ +
Glycosides - - - - - -
Saponins + ++ + + - +
Phenolics ++ +++ ++ + ++ +
Tannin + ++ + + + +
Carbohydrate + +++ + + + -
Protein ++ +++ + ++ +++ +

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Steroids and Sterols - - - - - -

+++ = Present in higher amounts, + = Present, ++ = Moderately present, - = absent

The various phytochemical compounds effect of processing of these nutritious rich seed.
detected are known to have beneficial importance Thus, the research focused on bringing to light their
in medicinal sciences. Alkaloids have been potentials for commercial exploitation.
associated with medicinal uses for centuries and
ACKNOWLEDGEMENTS
one of their common biological properties is their
cytotoxicity (Nobori et al., 1994). Several authors This study was financially supported by
have reported the analgesic (Antherden, 1969), Grant-in-Aid under Directorate of Extramural
(Harborne, 1973) antispasmodic and antibacterial Research & Intellectual Property Rights (ER&IPR),
properties of alkaloids (Stray, 1998; Okwu and Defence Research Development Organization
Okwu, 2004). Glycosides are known to lower the (DRDO), Ministry of Defence, Government of
blood pressure according to many reports (Nyarko India is gratefully acknowledged.
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