Antioxidant activity and determination of bioactive compounds by GC-MS in fruit methanol extracts-a comparative analysis of three Atalantia species from south India

Article history: Received on: 31/03/2015 Revised on: 08/09/2015 Accepted on: 12/10/2015 Available online: 27/02/2016 The members of genus Atalantia are widely used in Traditional, Folk and Ayurvedic systems of Medicine; of which three species are found in south India namely A. monophylla, A. racemosa, A. wightii. The fruits of these plants are picked and widely consumed by the tribal people. The present study aims to test the anti-oxidant potential of the methanol extracts of fruits and also to identify the bioactive compounds present in the extracts through GC/MS analysis. Among the three fruits tested for the antioxidant activity using the DPPH method, the crude methanolic extracts of A. monophylla shows higher potential. The IC50 value calculated was found to be 336.84 μg/ml for A. monophylla, 348.75 μg/ml for A. racemosa and 375.64 μg/ml for A. wightii. Ascorbic acid used as standard had an IC50 value of 103.03 μg/ml. Total antioxidant capacity was expressed as ascorbic acid equivalent and was calculated using the Phosphomolybdenum method. The methanol extract of A. monophylla, A. racemosa and A. wightii determined by phosphomolybdenum method exhibited significant antioxidant activity of 686mg/g, 592 mg/g and 655mg/g ascorbic acid equivalent respectively. Also the bioactive compounds in the Methanol extracts were evaluated using GC/MS.


INTRODUCTION
The genus Atalantia Correa (Family: Rutaceae) comprises of about 11 very closely related species (Swingle and Reece, 1967); of which three species are found in south India.Atalantia monophylla (Roxb.)DC. commonly called as wild lime is commonly seen in the dry evergreen forests of south India from the coastal regions to about 600m altitude.Atalantia racemosa Wight.(Bombay Atalantia) is seen in the evergreen forests of Peninsular India and Srilanka from about 150m to about 1000m.It is considered as a replacement taxon for A. monophylla.Atalantia wightii is endemic to shola forests of Western Ghats and is commonly referred to as Nilgiri Atalantia.It is distributed from 100m to about 1700m.
The members of the genus Atalantia are important plants in the understory layer of the forests.They are commonly found in the disturbed areas such as roadside, forest edges etc. and also along the sides of the streams and periphery of other water bodies where sufficient sunlight is available.
It is a source of food for larvae of several pollinators and also a source of food for seed dispersal agents (Vanitharani et al., 2011;Rajasekhar et al., 2006;Storz and Kunz, 1999;Punekar, 2002).Also the leaves of A. monophylla are an important source of food for the Asiatic elephant (Elephas maximus) (Mohapatra et al., 2013).Fruit, leaves and roots of the members of genus Atalantia are used for medicinal purposes.Traditional medicinal practitioners use the leaves of A. monophylla for the treatment of rheumatoid pain and glandular swelling.A decoction of the leaves is often applied for itching and other skin complaints (Panda, 2004) and also in treatment of dysentery (Jain, 1991).The leaves and roots are used as blood purifier and roots are given in cough and dysentery.Berries yield warm oil, which is valuable application in chronic rheumatism, paralytic limb and as stimulant (Krithikar and Basu, 1993;Panda, 2004).Fruit juice is anti-bilous and the fruit juice is consumed to treat stomach disorder by Kani tribals (Khare, 2007;John de britto and Mahesh, 2007).The juice pressed out from fruits heals endemic Cachexia (pitao) (Jain, 1991).Root is antispasmodic (Panda, 2004), antiseptic and stimulant and is used in treatment of snake bite (Jain, 1991;Krithikar and Basu, 1993) and antispasmodic pills are made from root (Jain, 1991;Manilal, 2003).
In addition to its medicinal value various parts of A. monophylla is used for several other purposes.Juice of berries is used for dyeing purposes.Wood is a good source of timber.It is also useful as a rootstock for breeding new cultivars of Citrus Linn (Guhabhakshi et al., 1999).Decoction of leaves of A. racemosa is used in the treatment of bronchitis, asthma and cough.The leaves are chewed to get relief from bronchitis.Leaf powder is used as blood purifier and decoction of the leaf is given every night as blood purifier.One tea spoon powder is given in cough and sour throat for 6 hours.Also a tea spoon of root powder is given every night as blood purifier (Pullaiah, 2006).The Kurichia tribes are using the leaf juice of A. racemosa Wight var.racemosa internally to treat acidity (Deviprasad et al., 2013).The ripe and unripe fruits are eaten by Paliyars (Arinathan et al., 2007).Also the fruits are pickled and eaten by several tribal groups such as Kurumba gounder, Sadaya gounder, Ariyan, Muthuvan and Kattunaikkan (Das et al., 2013).Kadars, Malasars and Muthuvan tribals of Anamalai hills use the whole plant of A. wightii to stupefy the fishes (Hosagoudar and Henry, 1996).Earlier works in A. wightii revealed the presence of Coumarins (Umbeliferone and geranyl umbeliferone) (Banerji et al., 1982) and few triterpenes (lupeol, lupenone, epi-friedelinol) (Desai et al., 1977).The fruits of the members of the genus Atalantia are picked and widely consumed by the tribal people.Therefore the present study aims to test the anti-oxidant potential of the methanol extracts of fruits and also to identify the bioactive compounds present in the extracts through GC/MS analysis.

Collection of plant material
The fruits and leaves were collected from different places in south India.A. monophylla samples were collected from Nagamalai hills of Madurai district, Tamil Nadu. A. racemosa and A. wightii were collected from Meghamalai hills, Theni, Tamil Nadu and the shola forests near Vaguvarai estate, Munnar, Kerala respectively (Fig. 1).The herbarium of the collected specimens were prepared and identified at Botanical Survey of India, Coimbatore.The herbarium specimen was deposited at the herbarium of department of Plant science, Madurai Kamaraj University.The samples were then shade dried and the moisture content of each sample was calculated.

DPPH radical scavenging assay
The DPPH reacts with methanol or ethanol to yield a purple color DPPH radical (DPPH).The presence of antioxidants which include polyphenolics and flavonoids in the sample will scavenge the formed DPPH radical and there by a decreased color will be absorbed which is spectrophotometrically measured at 517nm.To 0.5 ml of DPPH solution, add 2ml of the extract (100-1000g/ml) and the reaction mixture is vortexed for 10s and allow standing at room temperature for 30 minutes.The absorbance is recorded at 517 nm by using UV-Vis spectrophotometer.Compare with the 75% ethanol which acts as control solution.Ascorbic acid is used as reference antioxidant compound.

Phosphomolybdenum method
Antioxidant present in the sample reduce the Mo(VI) to Mo(V) which then react with the phosphate group sodium phosphate to form a green colored Mo(V) -Phosphate complex (Phosphomolybdenum complex) in an acetic medium.This complex is then spectrophotometrically measured at 695 nm.The tubes containing 0.2 ml of extract (100-1000μg/ml) is mixed with 1.8 ml of distilled water, 2ml of Phosphomolybdenum reagent solution.Incubate it at 95ºC for 90 minutes.The mixture is closed to room temperature and the absorbance is measured at 695 nm against reagent blank.The antioxidant capacity is expressed as Ascorbic Acid Equivalent (AAE).

GC/MS analysis of plant extracts
A Shimadzu QP-2010 plus with thermal desorption system TD 20 was used to obtain the chromatograms.The name and specification of the column used is Rtx ® -5MS (30m X 0.25 mm i.d.X 0.25 um film thickness).For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70 eV.Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1 ml/min, and an injection volume of 2 μl was employed (a split ratio of 10:1).The injector temperature was maintained at 260 °C, the ion-source temperature was 230 °C, the oven temperature was programmed from 100 °C (isothermal for 3 min), with an increase of 10 °C/min to 250 °C, then 20 °C/min to 380 °C, ending with a 19 min isothermal at 380 °C.Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 650 Da.The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.The components were identified based on the library search carried out using NIST and WILEY library.

DPPH radical scavenging activity
DPPH radical scavenging activity was tested to analyze the antioxidant potential of the methanol extracts of fruits of Atalantia species.Vitamin C (Ascorbic acid) was used as the standard and was compared with the sample for calculating the anti oxidant activity.Among the three fruits tested for the antioxidant activity using the DPPH method, the crude methanolic extracts of A. monophylla shows higher potential.All the three extracts show similar results.The percentage inhibition of methanolic extract of fruits was found to be 82.55%,72.4% and 80.04 at 1000μg concentration in A. monophylla, A. racemosa and A. wightii respectively (Fig. 2).Ascorbate used as standard showed 94.3 % inhibition at 1000μg concentration.The IC 50 value calculated was found to be 336.84μg/ml for A. monophylla, 348.75 μg/ml for A. racemosa and 375.64 μg/ml for A. wightii.
Ascorbic acid used as standard had an IC 50 value of 103.03 μg/ml (Table 1).

Phosphomolybdenum method
Total antioxidant capacity was expressed as ascorbic acid equivalent and was calculated using the Phosphomolybdenum method.The methanol extract of A. monophylla, A. racemosa and A. wightii exhibited significant antioxidant activity of 686mg/g, 592 mg/g and 655mg/g respectively (Table 2).The fruit of A. racemosa showed the maximum antioxidant activity among the three species.

GC-MS analysis of methanol extract of the three Atalantia species
The bioactive compounds in the Methanol extract of A. monophylla were evaluated using GC/MS.The analysis was done using Shimadzu QP-2010 plus with thermal desorption system TD 20, while the mass spectra of the compounds found in the extract was matched with the National Institute of Standards and Technology (NIST) library.Twenty seven compounds were identified from the mass spectra obtained.1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid was the major compound identified.In A. racemosa also 27 compounds were identified and n-Hexadecanoic acid was the major compound.In A. wightii 18 compounds were identified.β-Sitosterol was the major component in the methanolic extract of A. wightii.β-Sitosterol and Stigmasterol was present in all the three species (Table 3-5).
β-sitosterol is a very good for reducing the cholesterol levels in the blood and is sometimes used in treating hypercholesterolemia.The way of action is that β-Sitosterol inhibits cholesterol absorption in the intestinal absorption by competing thereby reducing the serum cholesterol levels (Matsuoka et al., 2008).It also shows anti-inflammatory and antipyretic activities (Gupta et al., 1980).Stigmasterol is used as a precursor for the manufacture of semisynthetic progesterone (Sundararaman and Djerassi, 1997).It is reported to be useful in prevention of ovarian, prostate, breast, and colon cancers (Ghosh et al., 2011).Stigmasterol also possesses anti-osteoarthritis and cholesterol lowering activity (Chen et al., 2012).

Table 1 :
IC50 values of DPPH radical scavenging activity of Methanol extracts of three species of Atalantia compared with the standard (Vitamin C).

Table 2 :
Total antioxidant capacity of three species of Atalantia by Phosphomolybdenum method.

Table 3 :
Compounds identified from the methanol extract of A. monophylla fruit using GC-MS.

Table 4 :
Compounds identified from the methanol extract of A. racemosa fruit using GC/MS.

Table 5 :
Compounds identified from the methanol extract of A. wightii fruit using GC/MS.