Isolation of bioactive molecule from Indian hog plum (Spondias mangifera Willd.) fruit

doi:10.21203/rs.3.rs-1970031/v1

Fresh Indian hog plum fruits (Spondias mangifera Willd.) were sliced, dried and powdered. Sequential extraction of this fruit powder was carried out using hexane, chloroform, ethylacetate and methanol. When these extracts were tested for different bioactivities, methanol extract showed the highest antioxidant activity and this extract was used to extract the bioactive molecule by silica gel column chromatography for isolating the pure bioactive molecule. The structure of the bioactive molecule was elucidated by analyzing UV, IR, LC-MS and 2D-HMQCT NMR spectral data and named it as Spondiol. This bioactive molecule showed an EC₅₀ value of 80, 104, 161 and 196 for DPPH radical scavenging activity, Superoxide radical scavenging activity, Lipid peroxidation inhibitory activity and metal chelating activity respectively. Spondiol also inhibited platelet aggregation with IC₅₀of 250 mg and 350 mg when induced by collagen and Adenosine Diphosphate (ADP) respectively. It also controlled the growth of bacteria viz., b. subtilis, M. luteus, S. fecalis, S. aureus, S. typhi, S. dysenteriae, K. pneumoniae and S. aureus.

Bioactive molecule

Spondias mangifera

Indian hog plum

fruit

Antioxidant activity

Platelet aggregation inhibitory activity

Antibacterial activity

Spondiol

Bioactivity

Fruits and vegetables are considered protective foods as they contain a sufficiently rich array of vitamins, minerals and various phytochemicals for important health processes to fight disease and strengthen the immune system. From time immemorial these have been used as traditional medicine and home remedies for various ailments and diseases. Unlike various synthetic chemicals, natural fruit products act as a safe, reliable and effective way to treat various ailments and their use in the food industry to delay oxidative damage caused by free radicals.

In India, a variety of fruits and vegetables are cultivated due to the prevalence of varied agroclimatic conditions. Some fruits are confined to certain geographical locations, which are exploited for the preparation of various products like pickles, juice, pulp and other culinary preparations. These underutilized fruits were found to have medicinal properties, the attributes of these were mentioned in the book of Ayurveda. The protective nature of these fruits is not only due to the presence of vitamins but also certain compounds responsible for a specific function in the body by scavenging free radicals (antioxidant), killing disease-causing microbes (antimicrobial), inhibiting platelet aggregation inhibition (antiplatelet). Thus, these compounds, which are called bioactive compounds, promote health of an individual with multiple functional properties with the least risk.

Keeping in view of the safety and multiple bioactive properties, plant extracts are used in traditional medicine for many ailments. This is evident from their free radical scavenging property or antioxidant activity.

Oxidative damage to the nucleic acids, proteins, and lipids by the free radicals leads to accelerated aging, various degenerative diseases viz., cancer, cardiovascular diseases, diabetes, etc. and other chain reactions in humans (Zhang et al., 2019; Maxwell, 1997). Antioxidant-rich fruits and vegetables scavenge free radicals produced during stress and pollutants reducing the risk of these diseases (Gupta et al., 2013; Sarin et al., 2012). The carcinogenic nature of synthetic antioxidants like BHA and BHT discouraged their use and prefer to use natural sources in food and medicine (Velioglu, et al., 1998). It reminds Hippocrates’ popular maxim “Let food be thy medicine and medicine be thy food”. Fruits and vegetables have fast emerged as “Healthy foods of the millennium”. They were also described as “Nutraceutical foods of the century,” owing to their health benefits. Nutraceutical properties of fruits and vegetables is attributed to the presence of phenolics, flavonoids and pigments.

Spondias mangifera Willd. commonly known as Indian Hog-Plum or Wild Mango belongs to the family Anacardiaceae originated in Tropical Asia (Campbell and Sauls, 1994). Anacardiaceae family consists of 8-12 species, which are widely spread in tropical regions of the world (De Souza et al. 1998). Spondias dulcis and Spondias mangifera grow abundantly in India (Basu and Rao, 1981; Ghosal and Thakur, 1981) while, S. purpurea and S. mombin are grow in Venezuela (Hoyos, 1989). Fruits of Spondias are popularly used as fresh fruit, pulp, juice and ice cream jelly, jams, pickles, relishes, soups, stews and juices (Morton, 1987) or canned (Ramsundar et al. 2002), used as flavour (Graham et al., 2004) medicines, ecological, industrial importance, gum exudates (Pinto et al., 2000; Pinto et al., 1996), additives in the food industry (Koubala et al. 2008) and Mushroom fructification.

The extensive work has been carried out on Spondias mombin and of course, Mangifera indica. But, Spondias mangifera is an untapped medicinal plant among various hog plums in the Mango family.

2.1. Fruit material

Fruits of India hog plum (Spondias mangifera Willd.) fruits were obtained from the local market. These fruits were washed thoroughly to remove dirt and debris followed by slicing and dried at 50^°C for 72 hours in a hot air oven and powdered to 60 mesh in a grinder (Apex Constructions, London).

2.2. Chemicals

HPLC grade Methanol, solvents like Hexane, Chloroform, Ethyl acetate and Methanol were obtained by Merck Limited, Mumbai. Silica gel (60-100 mesh and 100-200) was obtained from Qualigens Fine Chemicals, Mumbai. BHA, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), nicotinamide adenine dinucleotide (NADH) Trichloroaceticand acid (TCA) were purchased from Sigma (Sigma-Aldrich GmbH, Germany). Potassium ferricyanide, Ferrozine and Ferric Chloride were purchased from M/s Sisco Research Laboratories, Mumbai. Nitro blue tetrazolium (NBT), Phenazine methosulphate (PMS), Thiobarbituric acid (TBA) and Ethylene Diamine Tetra-Acetic acid (EDTA) were purchased from M/s Sigma Chemicals Co. (St. Louis, MO). Nutrient agar and Nutrient broth were obtained from HiMedia Laboratories Limited, Mumbai.

2.3 Platelet preparation

Samples were procured from healthy volunteers with substantial assurance that they were not administered any drugs during the two week period, prior to blood sampling. Blood was collected in buffered sodium citrate (3.8 % w/v), which is an anticoagulant with pH 6.5 at a ratio of 9:1 v/v and used within 3 hours of collection. Citrated blood was centrifuged at 1100 rpm for 20 min to produce platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was obtained by centrifuging the residual blood at 2500 rpm for 20 min. Platelet count was adjusted to 1.6 x 10⁷ platelets per ml of PRP.

2.4 Bacterial strains and inoculum preparation

The bacterial strains viz., Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus mirabilis, Yersinia enterocolitica, Micrococcus luteus, Staphylococcus aureus, Enterococcus fecalis, Bacillus subtilis, Bacillus cereus and Listeria monocytogenes, isolated from clinical samples were procuredfrom the Department of Microbiology, Mysore Medical College, Mysore, India. The antibacterial activity was tested against these microbes. Their morphological features and cultural characteristics were confirmed and also subjected to standard biochemical tests before experimentation (Krieg and Holt, 1984; Sneath et al., 1986). The test organisms were maintained on nutrient agar slants.

2.5. Isolation of bioactive compound from methanol extract

2.5.1. Preparation of extracts

Sequential extraction was executed using solvents of different polarities (from non-polar to polar). The compounds of different polarities were effectively and completely resolved using sequential extraction. About 100 g of different fruit powder was sequentially extracted using n-hexane, followed by chloroform, ethyl acetate, acetone and methanol at room temperature (25±2°C), at normal atmospheric pressure, by shaking at 100 rpm for 48 hrs. Each extract was filtered and concentrated by using a rotary evaporator (Buchi Rotavapor R-124, Switzerland). The concentrated extracts were freeze-dried and stored in a refrigerator until use. The antioxidant activity of methanol extract was very assuring and motivated for the isolation and purification the antioxidant compound from it.

2.5.2. Fractionation of the methanol extract

A glass column (450 x 450mm) was packed with activated silica gel (60-120 mesh) using n-hexane solvent. For large scale isolation of antioxidant compound, about 12 g of crude methanol extract was loaded and eluted stepwise with 500 ml of hexane, 500 ml of chloroform, 2000 ml of chloroform: ethyl acetate (90:10 to 0: 100, v/v) and 2500 ml of ethyl acetate: methanol (90: 10 to 0: 100, v/v). About 22 fractions measuring 250 ml each were collected and concentrated by using the rotary evaporator.

2.5.3. Thin Layer Chromatography (TLC)

Thin layer chromatography technique was used to isolate the volatile compounds. Aliquots of each concentrated fraction were loaded on the activated silica gel TLC plates (20 x 20 cm). The plates were developed using the following ratios viz., chloroform: ethyl acetate (80:20), chloroform: ethyl acetate (60:40), chloroform: ethyl acetate (50:50), chloroform: ethyl acetate (30:70), chloroform: ethyl acetate (25:75) and chloroform: ethyl acetate: methanol (15:75:10) solvents. TLC plates with the spots were located by exposing the plate to iodine fumes. Fractions were pooled into seven fractions (Fr.1- Fr.7) R_f values. Among the seven fractions, five fractions (Fr. 3 – Fr. 7) tested showed antioxidant activity.

2.5.4. Further purification of bioactive fraction

Antioxidant activity guided column chromatography was carried out for further purification of fraction 7 (Fr. 7). Eluting 3.60 g of Fr. 7 by 100 ml of chloroform followed by 1000 ml of chloroform: ethyl acetate (95:5 to 0: 100, v/v) and by 300 ml of ethyl acetate: methanol (90:10 to 0: 100, v/v) through 100-200 mesh silica gel with 450 x 20 mm column yielded 15 fractions of 100 ml each. These fractions were concentrated by a rotary evaporator for further loading of these aliquots on the TLC plates. Based on the R_fvalue, these fractions were pooled into five fractions (Fr. 7.1 – Fr. 7.5). These fractions were tested for antioxidant activity (Fig. 2c). Among these five factions, fraction 7.4 (Fr. 7.4) exhibited the highest antioxidant activity. Eluting 874 g of Fr. 7.4 with 100 ml of chloroform: ethyl acetate (90: 10 to 0: 100, v/v) followed by 400 ml ofethyl acetate: acetone (90: 10 to 0: 100 v/v) and 200 ml of acetone: methanol (90: 10to 0: 100, v/v) through100-200 mesh silica gel with 600 x 15 mm column yielded 20 fractions of 50 ml each. These fractions were concentrated by a rotary evaporator for further loading of these aliquots on the TLC plates. Based on the R_fvalue, these fractions were pooled into four fractions (Fr. 7.4.1 – Fr. 7.4.4).

Among these, sub-fraction three (Fr.7.4.3) obtained from third chromatographic step showed a single spot on TLC. This pure compound was subjected to various spectroscopic techniques for elucidation of the structure.

2.3.4. High performance liquid chromatography (HPLC)

The purified compound was tested for its purity using HPLC, using LC-10AT liquid chromatograph (Shimadzu, Singapore) equipped with C-18 column (300 x 4.6 mm, 5 m Thermo Hypersil) and methanol: water (60: 40) as a mobile phase with a flow rate of one ml min^-1. UV detection was carried out with a diode array detector (Shimadzu, Singapore).

2.4. Identification of bioactive compound

2.4.1. UV-Vis spectrophotometry

UV-Visible spectrum of the isolated compound was recorded on a Shimadzu UV-160A instrument (Shimadzu, Singapore) at room temperature. About 1 mg of isolated compound dissolved in 20 ml of methanol was used to record the spectrum (from 200-800 nm).

2.4.2. IR spectrometry

IR spectrum of isolated compound was recorded on a Perkin-Elmer FT-IR Spectrometer (Spectrum 2000) at room temperature. About 1 mg of isolated compound dissolved in 10 ml of DMSO was used to record the spectrum (frequencies between 4000 and 400 cm^-1).

2.4.3. Liquid chromatography- Mass spectrometry (LC-MS)

Mass spectrum of the isolated compound was recorded on instrument HP 1100 MSD series (Palo Alto, CA) by electro spray ionization (ESI) technique with a flow rate of 0.2 ml min^-1 on C-18 column and total run time of 40 min. Diode array was used as a detector. About 1 mg of isolated compound dissolved in five ml of methanol was used for recording the spectrum.

2.4.4. Two-Dimensional Heteronuclear Multiple Quantum Coherence Transfer Spectroscopy (2D-HMQCT) NMR spectra

NMR spectra were recorded on a Bruker DRX 500 NMR instrument (Rheinstetten, Germany) operating at 500 MHz for ¹H and 125 MHz for ¹³C at room temperature. A region from 0-12 ppm for ¹H and 0-200 ppm for ¹³C was employed. Signals were referred to internal standard tetramethylsilane. About 45 mg of isolated compound dissolved in 0.75 ml of DMSO was used for recording the spectra.

2.5. Antioxidant activity

2.5.1. DPPH free radical scavenging activity

DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity was determined according to the method described earlier (Blois, 1958; Bondet et al., 1997; Moon and Terao, 1998). The test samples (10- 100 ml) were mixed with 0.8 ml of Tris-HCl buffer (pH 7.4) to which 1 ml of DPPH (500 mM in ethanol) was added. The mixture was shaken vigorously and left to stand for 30 min. Absorbance of the resulting solution was measured at 517 nm in a UV-Visible Spectrophotometer (UV-160A, Shimadzu co. Japan). The radical scavenging activity was measured as a decrease in the absorbance of DPPH. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. Radical scavenging potential was expressed as EC₅₀value, which represents the sample concentration at which 50 % of the DPPH radicals scavenged.

2.5.2. Super oxide radical scavenging activity

The super oxide scavenging ability was assessed according to the method of Nishikimi, Rao, Yagi, (Nishikimi et al., 1972) with slight modifications. The reaction mixture contained NBT (0.1 mM) and NADH (0.1 mM) with or without sample to be assayed in a total volume of 1 ml of Tris-HCl buffer (0.02 M, pH 8.3). The reaction was started by adding PMS (10 mM) to the mixture, and change in the absorbance was recorded at 560 nm every 30 seconds for 2 min. The percent inhibition was calculated against a control without test sample. Radical scavenging potential was expressed as EC₅₀value, which represents the sample concentration at which 50 % of the radicals scavenged.

2.5.3. Lipid peroxidation inhibitory activity

Lipid peroxidation inhibitory activity was determined according to the method described earlier (Duh et al., 1999). In brief, egg lecithin (3 mg/ml phosphate buffer, pH 7.4) was sonicated in dr. Hielscher Gmb_H, UP 50H ultraschallprozessor (DrHielscher GmbH, Teltow, Berlin, Germany). The test samples (100 ml) were added to 1ml of liposome mixture, control was without test sample. Lipid peroxidation was induced by adding 10 ml FeCl₃ (400 mM) and 10 ml L-ascorbic acid (400 mM). After incubation for 1 hour at 37^°C, the reaction was stopped by adding 2 ml of 0.25 N HCl containing 15 % TCA and 0.375 % TBA and the reaction mixture was boiled for 15 min. then cooled, centrifuged and absorbance of the supernatant was measured at 532 nm. Inhibitory activity was expressed as EC₅₀value, which is sample concentration inhibited 50 % of lipid peroxidation.

2.5.4. Metal chelating activity

The chelation of ferrous ions by the test sample was estimated by the method described earlier (Decker and Welch, 1990; Dinis et al. 1994). Briefly, the test samples at different concentrations were added to a solution of 2 mM FeCl₂ (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml) and the mixture was vigorously shaken and left standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the mixture was read at 562 nm against a blank. EDTA was used as positive control. Results were expressed as EC₅₀value, which represents the sample concentration at which 50 % of metal chelation occurred.

2.6. Platelet-aggregation inhibitory activity

Aggregation was measured turbidimetrically at 37°C with constant stirring at 1000 rpm in a Chronolog Dual Channel Aggregometer. About 0.45 ml of PRP was kept stirred at 1200 rpm at 37°C, and aggregation was induced by collagen (10 mM) and ADP (10 mM). The change in turbidity was recorded with reference to PPP using an omniscribe recorder for at least 5 min. The slope was calculated and it was used as control. Similarly, 100-500 mM of the differentfruit extracts and isolated bioactive compounds were added to PRP, incubated for five min after which collagen (10 mM), was added. Platelet aggregation was recorded using an omniscribe recorder for 5 min. The slope was calculated. The difference in the slope between the control and the treated was expressed as percent inhibition of platelet aggregation by differentextracts.

2.7. Antibacterial activity

2.7.1. Minimum inhibitory concentration (MIC)

The minimal inhibitory concentration was determined according to the method described by Jones et al., 1985. A separate concentrate (20 ppm to 300 ppm) of a separate compound and 100 ofl bacterial suspension (105 CFU / ml) was applied aseptically in10 ml of the distinct broth and incubated. -24 h at 37 ° C. Growth was observed both by observation and by measurement O.D. at 600 nm at normal times followed by plating installation. The beating was performed by transferring the bacterial suspension (105 CFU / ml) to a sterile Petri plate and combined with a Nuttenent agar medium (HiMedia Laboratories Limited, Mumbai, India) was approved for intensification. Low concentration of the experimental sample that did not show significant growth was recorded as low inhibitory concentration. Three tube settings are kept in the test for each test sample. Amoxicillin (100 µg / mL) has been used as a positive control.

The minimum inhibitory concentration was determined according to the method described by Jones et al., 1985. Different concentrations (20 ppm to 300 ppm) of isolated compound and 100 ml of the bacterial suspension (10⁵ CFU/ml) was placed aseptically in10 ml of nutrient broth separately and incubated at 37^°C for 24 h. The growth was observed visually and measuring O.D. at 600 nm followed by pour plating. The plating was carried out by transferring bacterial suspension (10⁵ CFU/ml) to sterile petri plate and mixed with molten nutrient agar medium and allowed to solidify. The test sample with least concentration without visible growth was considered as the minimum inhibitory concentration. Three sets of tubes were maintained for each concentration of test sample. Amoxicillin (100 µg/mL) was used as a positive control.

2.7. Statistical Analysis

The experiments were carried out in triplicates. Significant differences (P < 0.05) were determined by Duncan’s Multiple Range Test (DMRT).

3.1. Isolation of bioactive compound

Hog plum fruit powder was subjected to extraction using solvents with increasing polarity. i.e., hexane followed by chloroform, ethyl acetate, acetone and finally methanol. From 100 g of hog plum fruit powder yielded 0.85g, 1.55g, 4.08g, 4.56g and 7.4g of hexane, chloroform, ethyl acetate, acetone and methanol extracts (Fig. 1). All the extracts were tested for their DPPH and super oxide radical scavenging activity. Among all the extracts, methanol extracts of Spondias mangifera cv. Hal exhibited highest DPPH and super oxide radical scavenging activity (Fig. 2b), hence, it was selected for the isolation of antioxidant compound. Silica gel column chromatography of methanol extract yielded 22 fractions. These fractions were loaded on the TLC plates and based on their R_f values, they were pooled into seven fractions from Fr.1 to Fr.7. All these fractions were evaluated for their DPPH radical scavenging activity, Fr. 7 recorded the highest activity of 51.8% (Fig. 2c). Subjecting Fr. 7 to silica gel column chromatography resulted in 15 fractions. These fractions were loaded on the TLC plates and based on their R_f values, they were pooled into five more sub-fractions from Fr.7.1 to Fr.7.5. Among these sub-fractions, Fr. 7.4 recorded the highest DPPH scavenging activity of 72.5% (Fig. 2c). Further purification of Fr. 7.4 was also carried out by silica gel column chromatography which resulted in 20 fractions.

These fractions were loaded on the TLC plates and based on their R_f values, they were pooled into four more sub-fractions from Fr.7.4.1-Fr.7.4.4. Among these, Fr. 7.4.3 exhibited highest DPPH radical scavenging activity respectively of 72.5% (Fig. 2c). The purity of Fr. 7.4.3 was confirmed by the TLC and by HPLC. Structure of the purified compound was elucidated by subjecting it to UV, IR, NMR and LC-MS.

3.2. Identification of bioactive compound

The structure of the bioactive compound was elucidated after analyzing the data obtained by various spectroscopic techniques. The molecular weight was determined using LC-MS. Mass spectrum showed the parent molecular ion at 371. The molecular formula of the compound was found to be C₁₈H₂₆O₈. The UV maxima were observed at 269 nm, 217 nm and 243 nm. Identification of specific functional groups was carried out using IR spectra. The O-H stretching observed at 3403 cm^-1 is the characteristic of hydrogen bonded -OH group. The stretching at 2996 cm^-1 and 1012 cm^-1 can be attributed to alkyl and C-O-C stretching vibrations respectively. The alkyl C-H bending vibration has been observed at 1427 cm^-1. (Table 1).

Table 1

Spectral data of the Spondiol.
Spectral	Spondiol
UV	269 nm, 217 nm and 243 nm
IR	3403 cm^− 1 (OH stretching) 2996 cm^− 1 (Alkyl -CH stretching) 1012 cm^− 1 (C-O-C stretching) 1427 cm^− 1 (C-H bending)
Mass	371 (M^+ 1)
NMR	Signal	¹³C (ppm)	¹H (ppm)
NMR	1-CH 2-CH 3-C 4-CH 5-C 6- C 7-CH 8-CH 9-CH 10-CH 11- CH₃ 14-OH 16-OH 18- CH₃ 21-CH 22-CHOH 23-CHOH 24-CHOH 26-CH 27- CH₂ 28-OH	69.2 38.0 151.4 101.9 133.3 136.7 101.0 158.7 129.1 130.6 12.0 - - 18.5 70.6 74.2 72.6 94.8 76.9 62.6 -	3.91 2.91 - 4.83 - - 6.5 5.3 5.43 5.37 2.05 4.14 15.0 2.21 3.67 3.71; 4.81 3.49; 4.81 5.41; 2.0 3.98 3.54 4.78

A Two-Dimensional Heteronuclear Quantum Coherence Transfer (2D-HMQCT) NMR spectrum was recorded along with 1-Dimensional ¹H and ¹³C NMR spectra, which gave a clear indication of the carbon skeleton of the compound (Table 1). The -CH₃ protons showed peaks in the region of 2.05–2.21 ppm. There are two -CH₃ groups and the corresponding ¹³C peaks were detected between 12.0-18.5 ppm. The cyclic -CH₂ and -CH protons exhibited peaks in the range 2.91–5.43 ppm and the corresponding ¹³C peaks have been observed between 38.0-129.1ppm. The one -CH₂ protons showed doublet at 3.54 ppm indicating clearly both the -CH₂ groups are attached to -CH carbon. The three -CH groups attached to -OH moiety showed a ¹H peak at 3.49–5.41 ppm and the corresponding ¹³C peak at 72.6–94.8 ppm. Based on these spectral data, the structure of the compound was deduced as (2R,3R,4R,5S,6R)-5-(5-dihydroxy-4-((E)-2-hydroxyvinyl)-3-methyl-6-((Z)-prop-1-enyl) cyclohexa-1,3-dienyloxy)-6-(hydroxymethyl) tetrahydro-2H-pyran-2,3,4-triol. The structure of the molecule was coined as a “Spondiol” (Fig. 4). Molecular formula of Spondiol deduced was C₁₈H₂₆O₈ with a molecular mass of 371. The compound has been reported for the first time from Spondias mangifera fruit and it is not reported from any other source.

3.3. Antioxidant activities of Spondias mangifera fruit extract and isolated compound

Various antioxidant assays like DPPH radical scavenging activity, super oxide radical scavenging activity, lipid peroxidation inhibitory activity and metal chelating activity were tested for spondiol and methanol extract to evaluate their antioxidant activity. BHA was used as a standard for antioxidant assay.

3.3.1. DPPH radical scavenging activity

The stable DPPH radical is predominantly followed method for quick evaluation of the antioxidant activity as it forms stable molecule by pairing with free radicals (Liu et al., 2018). All extracts of Spondias mangifera fruit showed considerable DPPH scavenging activity. Methanol extract exhibited the highest radical scavenging ability of 38.8%. Except, for hexane extract, with an increase in solvent polarity the antioxidant activity, also increased (Fig. 2a).

Isolated compound ‘Spondiol’ from the methanol extract of Spondias mangifera showed DPPH radical scavenging activity with an EC₅₀ of 80µg/ml (Table 2). The antioxidant activity of this compound may be attributed to the presence of -OH and C = O groups, as reported in structurally similar type of compounds (Chen and Ho, 1995; Nikolaos, et al., 2003).

Table 2

Antioxidant activity of Spondiol
Antioxidant activity	EC₅₀ value* (µg/ml)
Antioxidant activity	Spondiol	BHA
DPPH radical scavenging activity	80 ± 3.5 ^b	5 ± 0.8 ^a
Superoxide radical scavenging activity	104 ± 3.7 ^a	258 ± 2.4 ^b
Lipid peroxidation inhibitory activity	161 ± 2.8 ^b	94 ± 1.6 ^a
Metal chelating activity	196 ± 2.6 ^a	N.D.
* Each value represents mean of three different observations ± S.D.
Mean values with different superscripts (a, b and c) differ significantly at P < 0.05

3.3.2. Super oxide scavenging activity

The Superoxide radicals were successfully repelled by the methanol extract (Fig. 2b). The superoxide anion plays an important role in the formation of reactive oxygen species (ROS) such as hydrogen peroxide, hydrogen peroxide and hydroxyl radical which can cause oxidative damage to lipids, proteins and DNA (Pietta, 2000). The Methanol extract of Spondias mangifera cv. Hal not showed metal chelating activity and lipid peroxidation inhibitory activity.

Isolated compound ‘Spondiol’ from the methanol extract of Spondias mangifera showed superoxide radical scavenging activity with an IC₅₀ 104 µg/ml (Table 2). The activity was higher than that of BHA (258 µg/ml). Superoxide radicals are generated during the normal physiological process mainly in mitochondria. Although superoxide anion is by itself a weak oxidant, it gives rise to the powerful and dangerous hydroxyl radicals as well as singlet oxygen both of which contribute to oxidative stress (Meyer and Isaksen, 1995). Therefore, superoxide radical scavenging by antioxidants has physiological implications.

3.3.3. Lipid peroxidation Inhibitory activity

Lipid peroxidation is a free radical mediated propagation of oxidative insult to polyunsaturated fatty acids involving several types of free radicals. Its termination occurs in the biological system through enzymatic means or by radical scavenging activity by antioxidants (Heim et al., 2002). Spondiol showed lipid peroxidation inhibitory activity with an IC₅₀ of 161 µg/ml (Table 2). Hydroxyl radical and perferryl ions are highly reactive and act as the actual initiating species for cellular lipid peroxidation (Fridovich, 1989).

3.3.4. Metal chelating activity

Isolated compound, spondiol exhibited bioactivity of metal chelating with an IC₅₀ of 196 µg/ml (Table 2). In Fenton and Haber-Weiss reaction, Iron is responsible for generating free radicals (Halliwell and Gutteridge, 1990). Oxidative damage is caused by the metal ion is a result of the production of oxyradical. This bioactivity plays an important role as it reduces the concentration of catalyzed transition metal in lipid peroxidation (Duh et al., 1999). This is the reason for the natural antioxidant activity of Spondiol.

3.4 Platelet-aggregation Inhibitory Activity

Spondiol showed platelet-aggregation inhibitory activity with an IC₅₀ of 250 µg and 350 µg against collagen and ADP respectively. Glycoprotein IV (CD 36) present on platelets plays an important role by acting as a specific receptor in the early stages of aggregation (Tandon et al., 1989). Another receptor Glycoprotein VI has also been identified under flow condition specifically again for induced aggregation (Moroi, 2004). Spondiol concentration of IC₅₀ for collagen and ADP were used to investigate the effect of an increase in the duration of incubation from 1 to 2 and 4 minutes (Table 3). It was observed that as the incubation time doubled the percentage inhibition increased. This showed that the longer the contact with the spondiol, the better the binding to the surface glycoproteins.

Table 3

Effect of incubation time at IC50 of Spondiol on platelet-aggregation inhibitory activity.
Inducer	Amount IC₅₀ * (mg)	Per cent Inhibition Incubation Period
Inducer	Amount IC₅₀ * (mg)	1 min	2 min	4 min
Collagen	0.250	51.2 ± 2.1^a	79.2 ± 2.1 ^c	85.5 ± 2.4 ^e
ADP	0.350	49.6 ± 1.9 ^a	64.5 ± 1.4 ^b	76.7 ± 2.7 ^d
* Each value represents mean of three different observations ± S.D.
Mean values with different superscripts (a, b, c, d and e) differ significantly at P < 0.05

Regulation of platelet activity by using specific pharmacological agents has proven to be a successful strategy for the prevention of thrombosis. Platelet-aggregation inhibitory agents, such as aspirin, dipyridamole, thienopyridines, and platelet glycoprotein IIb/IIIa antagonists have amply demonstrated their utility in preventing and treating coronary artery thrombosis (Van De and Steinhubl, 2000). Results obtained by the incubation of human or animal platelets with isolated polyphenols suggest that the platelet-aggregation inhibitory properties may be attributed to the inhibition of TxA2 formation (You, et al., 1999), thromboxane receptor antagonism (Hubbart et al., 2003), protein kinase C activation (Ganet-Payrastre et al., 1999) and phosphoinositide synthesis.

3.4. Antibacterial activity

3.4.1. Minimum inhibitory concentration

Spondiol exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria. It was more effective against wide spectrum of bacteria viz. M. luteus, S. aureus, B. subtilis, S. typhi, S. dysenteriae, S. fecalis, and K. pneumonia. The most striking increase in activity was observed against M. luteus, S. aureus, B. subtilis and S. typhi with MIC of 50, 60, 70 and 80 ppm respectively. It also inhibited the growth of five Gram-negative bacteria viz. S. typhi, K. pneumoniae and S. dysenteriae (Table 4).

Table 4

Antibacterial activity of Spondiol.
Bacteria	Spondiol
Bacteria	MIC (ppm) *
B. subtilis	70
M. luteus	50
S. faecalis	110
S. aureus	60
S. typhi	80
K. pneumoniae	140
S. dysenteriae	100
*Each value is a mean of three different observations.

However, these were not inhibited by the methanol extract. High antibacterial activity of spondiol for wide range of bacteria may be attributed to its structural components. It possesses two-hydroxyl and two-methyl along with an aromatic ring.

Enhanced antimicrobial activity of spondiol is due to the presence of an aromatic ring which are capable of showing electron delocalization (Feixas et al. 2015; Ultee et al. 2002). Similar antimicrobial bioactivity was also reported in several other compounds like capsaicin, caffeic acid, carvacrol, eugenol and methanol (Basu et al. 1996; Ali-shtayeh et al. 1997; Cowan, 1999).

Multiple bioactive compounds were successfully isolated and characterized from Spondias mangifera, viz. ‘Glycospondin’ from ethyl acetate extract and ‘Spondiol’ from methanol extract. These compounds were found to be reported first time from the Spondias mangifera fruit. They are not reported previously from any other sources. ‘Glycospondin’ exhibited high platelet-aggregation inhibitory activity while ‘Spondiol’ showed high antioxidant activity. However, both the bioactive compounds exhibited multiple bioactivities. ‘Spondiol’ was used as a biomarker for the determination of maturity of Spondias mangifera during fruit growth and development (Sivaprasad et al., 2011).

Spondiol was isolated by sequential extraction of Spondias mangifera fruits followed by bioactivity-guided fractionation with silica gel column chromatography and characterized by different spectral analysis. Spondiol is a novel compound not reported previously from any other source. It exhibited a multi system antioxidant activity, platelet-aggregation inhibitory activity and also antimicrobial activities. The antioxidant activity of spondiol includes reductive ability, metal chelator, hydrogen donating ability and scavengers of super oxide radicals. Spondiol also showed its anti-bacterial activity on some of the tested bacteria. Antioxidant, platelet-aggregation inhibitory activity and antibacterial activity shown by spondiol need to be exploited.

Acknowledgement

We are thankful to Dr. V. Prakash, Director, CFTRI, Mysore for his keen interest in the work and encouragement. Mr. M. Sivaprasad thanks the CSIR, New Delhi, India, for awarding the Research Fellowship. We also thank Sophisticated Instruments Facility, IISc, Bangalore, for the NMR analysis and Molecular Biophysics Unit, IISc, Bangalore for the LC-MS analysis.

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No competing interests reported.

Isolation of bioactive molecule from Indian hog plum (Spondias mangifera Willd.) fruit

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

3. Results And Discussion