Antioxidant Activities of Chloroform and Aqueous

Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195 Available online at www.jacsonline.org

ISSN: 2089-6328 Research Article

Antioxidant Activities of Chloroform and Aqueous Fractions of Myrmecodia Pendenss Extract: A Preliminary Study Yohanes Buang*, Ermelinda Noya, Pius D. Ola, and Theo Da Cunha Department of Chemistry Faculty of Science and Engineering, Nusa Cendana University ABSTRACT There have been elucidated the antioxidant activities of chloroform and aqueous fractions of methanol extracts isolated from myrmecodia pendenss. The study was firstly started by maceration of myrmecodia pendenss powder in certain quantity of pure methanol solvent and followed by gradual partition of the methanol extract with chloroform and distillated water and then purified with column chromatography. The chemical contents migrated into both those later solvents were detected by GC-MS and their functional groups were detected by FT-IR instruments. Thereafter, the antioxidant activities of the migrated compounds in each those solvents were determined using hydrogen peroxide protocol and the absorbance was measured at 240 nm. The ascorbic acid solution was used as control. The results showed that FT-IR spectrum showed methylene and ethene groups are existed within the compounds contained by the extracts as well as the hydroxyl, carbonyl, aromatic, and keton groups. Furthermore, the GC-MS spectrum indicated the compounds that are existed within the chloroform and the aqueous fractions of the extract. The average absorbances of compounds contained within the chloroform and aqueous fractions were 0.161 and 0.112, respectively, whereas that of the control was 0.085. Those absorbance data conclusively indicated that higher absorbance higher residue of chromophore content existed within the fraction and higher failures of hydroxylation reactions. It was however, the antioxidant activities of the aqueous fractions were higher than those of the chloroform fractions. Keywords: antioxidant, myrmecodia pendenss, chromatography, UV-VIS spectrophotometry, GC-MS *Corresponding author: [email protected]

hydrogenations. Both those possible reactions could reduce amounts and therefore the effects Antioxidants are electron donating atom of the free radicals. or molecule that could ameliorate the negative The chemical compounds that act as impact of free radicals. Antioxidants are capable antioxidants are the class of numerous comof removing, cleaning, resist or counteract the pounds including phenols such as phenolic and effects of the formation of reactive oxygen polyphenolic. The class and amounts of those species (Uchida et al. 1999, Khodr and Khalil antioxidant compounds are widely available in 2001, Lee et al. 2008, and Buang et al. 2011). nature, especially in plants. Antioxidants are substances that are known to The myrmecodia pendenss is a plant neutralize the free radicals or materials which member of family Rubiaceae, consisting of 5 can prevent the body's biological systems from genera but only two genera are closest the adverse effects arising from the process or association with the ants and hydnophytum reaction that causes excessive oxidation. In the myrmecodia, however the plant function use as a availability of antioxidants within the body is medicine may only be hydnophytum and inadequate, both those provided by the body myrmecodia pendenss. The myrmecodia systems and from the externals such as such as pendenss (Fig. 1) has been known to contain vitamin E, vitamin C, carotenoids, etc. the active antioxidant compounds (Hertiani et al. immune system would probably decrease and 2010). The greatest diversity of plants is found cause premature aging and the onset of various in Papua, people often say these plants diseases. Various possible reaction mechanisms originated from Papua due to a local high plains in biocellular system involve hydroxylations and species specific. The local society use J Applied Chem. Sci. 2013, Vol. 2 Issue 1 187 | P a g e

Introduction

Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195 myrmecodia pendenss extracts as traditional medicine, which is empirically proven efficacious to cure various diseases naturally and safely. Parts of the plant used as medicine is meat hypocothyl (caudex). On the inside part of the caudex, hundreds of ants inhabited the hypocothyl labyrinth. The extracts of this plants are also known to have benefits for different types of tumors and cancers as wel as effective for curing various gout and blood circulation (Natural, 2006). Several studies have shown that coudex water raction has antioxidant activity with 37.03 mg/mL of LC50 value.

(a)

(b)

Fig. 1. Myrmecodia pendenss from Belu District, before cleaned (a) and the caudex (b)

The mymercodia pendens plants also spread in NTT province such as in Sumba, Manggarai, Ende, South and North Middle Timor districts, and areas located at altitudes of 600m from surface lowlands, including Belu district. Belu Regency society in general use mymercodia pendens extracts as traditional medicines to cure various diseases such as: blood circulation, gout and other diseases. Various in climates, dry and rainy seasons, soil contents, etc. could differentiate chemical compositions of plants as well as the mymercodia pendens. Therefore, to determine whether the mymercodia pendens extract from Belu district has antioxidant activity compounds, it is necessary to isolate, identify, and determine the antioxidant activity of the compounds containing within the extracts.

Material dan Methods The mymercodia pendens sample used in this study was taken from Desa Haekesak, Kab. Belu. NTT. The chemicals include ethyl acetate, methanol, distillated water (DW), chloroform, formic acid, benzene, ethanol, ammonia, H2O2, petroleum ether (PE), etc. The equipment used Page | 188

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involved preparative thin layer plate (TLC) and column chromatography, GC-MS, and UV-Vis spectrophotometer. a. Sample preparation Cleaned the outer shell of the caudex, then take as much as 3 kg, washed thoroughly to remove impurities and allowed to dry at room temperature. Once dry the samples grinded. The powder obtained was weighed. b. Maceration Method Caudex powder weighed 100 grams and put into erlenmeyer 2L size and then 1000 ml of methanol was added and stirred until all the caudex powder immersed within methanol. Erlenmeyer tightly plugged with rubber and left for overnight/24 hours. The mixture was filtered with whatman 42 filter papers. Obtained filtrate evaporated at a temperature of 550C with a rotary evapo-rator, to obtain a viscous solution. The viscous solution obtained was then stored in sealed bottles. c. Partition Method Weighed 2.5 grams of viscous solution extract, then add 10 ml distilled water, digested until homogeneous. The above solution was transferred into a separating funnel (rinse residue with DW to a final volume of 20 mL). Inserted successively 25 mL chloroform and allow few times until partition clearly performed and then separated the chloroform fraction. Repeated the protocol for 4 times and collected all chloroform fractions in a new erlenmeyer. Removed the DW residue and kept as aqueous fraction. Those partition results were evaporated on a rotary evaporator, and kept evaporated results as the chloroform and the aqueous fraction, respectively. d. Thin layer chromatography Method Taken 2.0 grams of chloroform extract partition results and dissolved in 25ml PE. Spotted on a TLC plate with a capillary tube and then eluted with 3 type mobile phase below: 1. Ethyl acetate: methanol : DW = 10 : 1.35 : 1 2. Chloroform:ethyl acetate: formic acid = 10 : 8 : 2 3. Benzene : ethanol : amonia = 18 : 2 : 0.2

Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195

Fig. 2. Standard absorbance and standard curve (a) and antioxidant activities of the extracts (b)

The separated spots detected with UV light or with dyes, I2. The band style was recorded by photographed. These results were used to obtain the best mobile phase composition to prepare column/ separation. e. Chromatography column Method Used glass column with a diameter of 1.6 cm and a length of ± 25 cm. A total of 2 grams of chloroform extract partition results was inserted into the column and eluted with the best mobile phase found, then evaporated column fractions and further determine the chemical composition using GC-MS and determination of the antioxidant activity. f. Antioxidant activity Weighed each sample fraction of 25.0 mg and diluted with DW in 100 mL flask and filled. Into each 15 mL of each sample extract was added 10 mL H2O2 900 ppm. The mixture was homogenized thoroughly and left for 30 minutes in dark place; the absorbance was measured using UV-Vis spectrophotometer at 240 nm. The same protocol was performed for vitamin C as a positive control.

Results and Discussion In this study, the myrmecodia pendenss extract was used, its caudex known to have therapeutic effect that can reduce or ameliorate various degenerative diseases such as previously mentioned. The extract is currently known as the

traditional medicine of people in original source of the myrmecodia pendenss. a. Antioxidant activity The maximum wavelength of solution absorbance was found at 240 nm. The wavelength 240 nm was used to determine the absorbance of the H2O2 standard solution, with concentration of 500-900 ppm (Fig. 2a), and the sample treated as well as the control (Fig. 2b). The standard absorbances, calibration curve, and the absorbances of the treated samples and the control are reported in Fig. 2a-b. In this study, the antioxidant activity of the extracts was reported as the absorbances. The differentiation in absorbances reported by the UV-Vis instrument are possible to be interpretated. The absorbance of chloroform fraction was higher than those of by the two others, the Vitamin C and Aqueous groups, indicated that the antioxidant activities of the compound existed within the chloroform fraction was lower than those other two groups. The antioxidant effects reported by the H2O2 protocol occur through hydroxylation reactions. This was because the H2O2 molecule being interacted with UV energy and liberates two *OH radicals with is respected with the Lewis structure Fig. 3). The yielded *OH would further hydroxylate the chomophore groups on targeted molecule being energized by the UV light. The chromophore groups of the targeted molecule

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Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195 being energized with UV cause their π bonds shifted into *π and therefore hydroxylation reactions are possible to occur accordingly. The suggested mechanisms are reported in Fig. 3a-b. Reading the absorbance data reported in Fig. 2 that the higher absorbance conclusively indicates the amounts of the chromospheres groups larger than those lower absorbances. In case, the unsaturated bonds (π bonds) were failed to reach the *π transition, once activated energy terminated, those bonds moved back to the ground state (at initial position) as π bonds. Consequently, the presence of the *OH from the H2O2 molecule was failed to perform the hydroxylation reactions. Hence, higher absorbance recorded by the UV-Vis instrument indicates less hydroxylation occurred and vise versa. The higher absorbance performed by the chloroform fraction of the mymercodia pendens extract than those two others therefore indicated that the antioxidant effects of the targeted compounds existed within the chloroform extract of the mymercodia pendens lower than those others. The added H2O2 into reaction system liberated *OH radicals and shift those π bonds of the antioxidant compounds into the σ bonds through the hydroxylation reactions, the antioxidant compounds absorb the *OH radical from the H2O2 molecules. The mechanism of the σ bonds breakings are reported in Fig. 3. Overall, higher antioxidant effects of the extracts, such as mymercodia pendens extracts, higher their

(a)

hydroxylation level accompanied with lower their absorbance and vise versa. The data shown in Fig. 2b indicated that the absorbance of vitamin C, the aqueous and the chloroform fractions of the mymercodia pendens extracts were 0.085, 0.112, and 0.161, respectively. Those data said that antioxidant activities of Vit. C > aqueous fraction > chloroform fractions of the mymercodia pendens extracts.

b. The infra red spectrums and the types of compounds of the chloroform fractions The infra red spectrums of the compounds existed within chloroform fractions of the extracts are reported in Fig. 4a. The vertical scale represent the quantitative measures which is reported in percent transmission, while the horizontal scale represent the qualitative measures of each functional group contained by the sample that is reported as wave number (cm-1) or frequency. Lower percent transmission higher the absorption and therefore percent transmission represents the densities of the functional group. The wave number at the minimum point of the transmission is used to identify the group. Numbers of identical groups in a molecule alter the relative strengths of the absorption in a spectrum. For example, a single C—H absorption is relatively weak, but if a compound has many C—H bonds with their vibrations similarly, the collective effects of C—H absorptions generate a peak that is medium or even strong.

UV

Abs-240nm

Radicalisation of H2 O2 molecule

H-O

O-H

*OH

R

(b)

UV +

R

H-O

*OH

O-H

*OH

R

*OH

R

+

OH

*OH + *OH OH

Targeted antioxidant molecule being energized by UV light

Radicalisation of the targeted antioxidant molecule

Hydroxylated antioxidant

Fig. 3. The radicalization of H2O2 and hydroxylation of the antioxidant molecules

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Fig.4. IR spectrum and The TIC data of compounds contained in CHCl3 fractions


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Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195 Occasionally, a weaker band undergoes overlapping with a stronger one; this is called shoulder (Fessenden et al. 1991). The FT-IR data reported in Fig. 4a indicates that the chloroform fraction of the extracts contain 7 different functional groups that are appeared two peaks at the stretching vibrations and seven peaks at finger print areas. The functional group found at 3448.49 cm-1 indicates the stretching vibrations of the O-H groups. The wide band shown indicates bonded hydrogen that occurs among the O-H group existed with the compounds. The bending vibration of the O-H group presumably found at 1323.08 cm-1. Yet, the presence of the peak at 1629.74 cm-1, N-H bending vibrations, might indicate that the N-H stretching also existed and mixed with those O-H groups that were appeared at 3448.49 cm-1. The C=N and C=C stretching vibrations are also possible to be shown by the 1629.74 cm-1. The peaks appeared at the 1402.15 cm-1 strongly indicates the C-H bending vibrations. Therefore, the functional groups shown at the 3448.49 cm-1 were not only mixture of the O-H and the N-H groups but also existed together within the C-H groups. The peaks shown at 1126.35 and 1193.85 cm-1 strongly indicates the C-O, C-C, and C-N stretching vibrations. The peaks shown at 472.53cm-1 indicated the weak stretching vibrations of the S-S group, while those peaks shown at the 619.11 and 651.99 cm-1 indicated the stretching vibrations of the C-S groups. Those qualitative interpretations for the infra red spectrums of the mymercodia pendens extracts were in agreements with of the IR spectrum data reported in various literatures (Fessenden 1981, Silverstein et al. 1991, Wade, 1991, and Morrison et al. 1992). Overall, the compounds contained within the extracts have these OH, C=C, C—H, C—N, C—O, C—S, and S—S functional groups. Although those infra red data could not inform the molecular structure of the compounds contained, but these functional groups could be used to compare with the other data, such as the Page | 192

data provided by the GC-MS instruments. This study was continued with GC-MS instrument to have further information regarding the molecular compound contained by the extract although without separation of each compound contained within the extract. This is also therefore the antioxidant activities reported previously (Fig. 2) indicated the simultaneous activities of the compounds contained within the extracts. The compounds contained within the chloroform extracts were detected by the total ion chromatography (TIC) detector (GC instrument) and the TIC chromatogram are reported in Fig. 4, a TIC detector is known to be capable to detect all compounds regardless of the elements involved as reported in the literature of the GC instrument. As shown in Fig. 4, there are 10 compounds detected from the chloroform fraction of the extracts. Although the TIC data (Fig. 4b) showed 10 compounds, but among these compounds which have high contents only were discussed in this report. Regarding to these TIC spectrums, the peak 6, 7, and 8 are discussed because of there are providing relative concentrations higher than the others. Those are the dominant compounds contained within the chloroform fraction of the extracts. a. The compounds reported on peak no. 6 The GC-MS analyses for peak no. 6 provided spectrums as reported in Fig. 5. The based peak, the highest relative abundance (100%) among the spectrums, of the peak no. 6 has molecular ion weight of 57 g/mol. This is the most abundant molecular ions among the stable fragments yielded from the peak 6. This is the highest abundance among the fragments. The other molecular ion peaks had lower abundance which was involved other 10 molecular ion fragments recorded. These are with the molecular weights of 207, 185, 129, 116, 98, 84, 71, 57(base peak), 43, 41, and 40 g/mol, respectively. Among those fragments, the less abundance was found at fragment with the molecular weight (MW) of 207 g/mol.


Reference-1 Noya EF et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195

Sample TIC –Peak 6

Reference-2

Reference-3

Reference-1

Fig.5. The mass spectra of the peak no. 6 and the 3 possible references

According to the reference data, the molecular ions reported in peak Reference-2 6 of GC-MS diagrams (TIC data) derived from reactive molecules that has MW of 569 g/mol (reference 1 hit). This decision was taken because, based on all available references, the similarity index (SI) of the compound existed within the peak 6 was suitable with reference 1, in whichReference-3 it has highest SI among all those three other possible references. Although, those three references show the similarity pattern of the fragmentation with the peak no. 6 compound but the eference-1 was most closely to the compound in extract with the similarity of 85% and therefore it could be estimated that the compound existed within the peak 6 has MW of 569 g/mol with the molecular formula C35H68O5 and its structure (Reference-1) is as reported in Fig. 6.

Fig. 6. The structure of hexadecanoic acid,1 hydroxymethyl,1,2-ethyl ester compound

These results were confirmable with the presence of the OH, C-H (of -CH2 and -CH3), C-O, and C=O groups that was reported and discussed in

part of infra re spectrum data. Therefore, based on the similarity index and the suitable functional groups existing in the FT-IR spectrums, the molecular structure of peak 6 is possible to the TIC peak of the hexadecanoic acid,1hydroxymethyl,1,2-ethyl ester compound. This simple estimation is still required to be compared with spectrums of its pure compound. b. The compounds reported on peak no. 7 The based peak of the peak no. 7 has molecular ion weight of 129 g/mol. This is the most abundance among the molecular ion fragments yielded from the peak 7. The other stable molecular ion peaks recorded by the instrument involved other 11 molecular ion fragments. Those are with molecular weights of 259, 241, 207, 147, 129, 111, 84, 70, 57, 55, 41, and 40 g/mol, respectively. The mass spectra of the compound present in peak 7 are reported in Fig. 7. According to the reference data, the molecular ions reported in peak 7 of GC-MS diagrams (TIC data) derived from reactive molecules that has MW of 370 g/mol (reference 1 hit). This decision was taken because, based on all available references, the SI of the compound existed within the peak 7 was suitable with reference 1 because it has highest SI among all those three possible references. Although, its


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Buang Y, et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195 similarity is only 96% but it could be estimated that the compound existed within the peak 7 has MW of 370 g/mol with the molecular formula of C22H42O4 and its structure is as reported in Fig. 8.

Fig. 8. The structure of hexadionic acid diethyl ester

These results were confirmable with the presence of the C-H (of -CH2 and -CH3), C-O, and C=O (1500-1900 cm-1) groups in the FT-IR spectrums shown in Fig. 4a. Therefore, based on the similarity index and the suitable functional groups existing in the FT-IR spectrums, the molecular structure of the compound reported by the peak 7 is possible to the hexadionic acid, diethyl ester compound. This simple estimation is still required to be compared with spectrums of its pure compound.

c. The compounds reported on peak no. 8 The based peak of the peak no. 8 has molecular ion weight of 55 g/mol. This is the most abundance among the molecular ions fragments yielded from the peak 8. The other stable molecular ion peaks recorded by the instrument involved other 14 molecular ion fragments. Those are with molecular weights of 281, 253, 207, 185, 171, 151, 129, 116, 98, 95, 81, 67, 55, 41, and 40 g/mol, respectively. The mass spectra of the compound present in peak 8 are reported in Fig. 10. According to the reference data, the molecular ions reported in peak 8 of GC-MS diagrams (TIC data) derived from reactive molecules that has MW of 266 g/mol (reference 1 hit). This decision was taken because, based on all available references, the SI of the compound existed within the peak 8 was suitable with reference 1 because it has highest SI among all those three possible references.


Although, there is only 86% its similarity but it could be estimated that the compound existed within the peak 8 has MW of 266 g/mol with the molecular formula C18H34O and its structure is as reported in Fig. 9. These results were confirmable with the presence of the C-H (of -CH2 and -CH3), C=C, and C=O groups in the FT-IR spectrums shown in Fig. 4a. Therefore, based on the similarity index and the suitable functional groups existing in the FT-IR spectrums, the molecular Page | 194


structure of the peak 8 is possible to the TIC peak of the 9-octadiene compound. This simple estimation is still required to be compared with spectrums of its pure compound.

Fig. 9. The structure of 9-octadiene compound


Reference-1

Noya EF et al./Journal of Applied Chemical Science 2013, VoL. 2 Issue 1: 187-195


Reference-1

Reference-2

Reference-3


tuberosa and Myrmecodia pendenss. OnLine Journal of Biological Sciences 10 (3): 136Based on the results of this study, the Reference-2 caudex 141, 2010 extracts of the myrmecodia pendenss that was Lee SJ, Seo KW, Yun MR, Bae SS, Lee WS, Hong KW, and Kim CD. 4-Hydroxynonenal originally from Belu District/Timor Island enhances MMP-2 production in vascular contains antioxidant compounds. The antioxidant smooth muscle cells via mitochondrial ROSactivities of the water extracts were higher than mediated activation of the Akt/NF-�B the chloroform fractions. Although the antioxidant signaling pathways. Free Radic Biol Med. 2008, 45: 1487. activities of the chloroform fraction is lower than Reference-3 Khodr B and Khalil Z. Modulation of inflammation by those water fractions but it might support that the reactive oxygen species: implications for caudex of the myrmecodia pendenss are aging and tissue repair. Free Radical Biology and Med. 2001, 30 (1): 1.Morrison RT and economically better as herbal medicine for the Boyd RN, 1992. Organic Chemistry, pp 590society. The present study, although failed to 596, 6th Ed. ISBN: 10987654321 identify all compounds contained in chloroform Natural. 2006. Senyawa Aktif Bersarang di Sarang fractions and even the water fraction of the Semut. Majalah : Jakarta extract, but this study at least provides preliminary Sidik. 1997. Antioksidan Alami Asal Tumbuhan, jurusan farmasi F-MIPA Universitas data pertaining to herbal medicine using caudex Padjajaran : Bandung extracts of the myrmecodia pendenss. Silverstein RM, Bassler GC, and Morril TC, 1991. Spectrometric Identification of Organic References Compounds, pp 91-164, 5th Ed. ISBN: 0-13Buang Y, Nagao K, and Yanagita T. 2011. Shochu 630179-7 Kasu against Fatty Liver Disease: The Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, therapeutic properties from basic science to and Osawa T. Activation of stress signaling clinical practice. LAP Lambert Academic pathways by the end product of lipid Publishing, Germany; ISBN: 978-3-8443-9730-7 Available online at www.amazon.com peroxidation. 4- Hydroxy-2-nonenal is a Fessenden RJ and Fessenden JS, 1981. Organic potential inducer of intracellular peroxide Chemistry, pp 318-330, Student Ed. ISBN: 0production. J Biol Chem . 1999, 274: 2234. 534-98024-4 Wade LG, 1991. Organic Chemistry, pp 444-489, 2nd Hertiani T, Sasmito E, Sumardi, and Ulfah M. Ed. ISBN: 0-13-642588-4 Preliminary Study on Immunomodulatory Effect of Sarang-Semut Tubers Myrmecodia

Conclusion

Source of support: Nil, Conflict of interest: None Declared J Applied Chem. Sci. 2013, Vol. 2 Issue 1

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