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Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria
j o u r n a l o f p h a r m a c y r e s e a r c h x x x ( 2 0 1 3 ) 1 e5
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria Julius K. Adesanwo*, Oluwatoyin O. Makinde, Craig A. Obafemi Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
article info
abstract
Article history:
Aim: This study was designed to assess the antioxidant property of crude methanolic
Received 29 July 2013
extract of Tetracera potatoria roots (MeTp); a bioactive constituent isolated from it, betulinic
Accepted 2 September 2013
acid (BA) and a combination of BA with ascorbic acid.
Available online xxx
Methods: The crude extract was obtained by cold extraction. Phytochemical screening of the crude extract was carried out using standard methods. Isolation of betulinic acid (BA) was
Keywords:
effected by Accelerated Gradient Chromatography (AGC) fractionation of MeTp. Structural
Tetracera potatoria
elucidation of the isolated BA was done with 1H and
Antioxidant activity
MeTp, isolated BA and BA plus ascorbic acid mixture (1:1) were evaluated for their anti-
Ascorbic acid
oxidant activity by 1,1-diphenyl-2-picrylhydrazyl, (DPPH) method.
Betulinic acid
Results: The phytochemical analysis showed the presence of alkaloids, flavonoids, cardiac
1,1-Diphenyl-2-picrylhydrazyl
glycosides, saponins and tannins. Anthraquinones were absent. The 1H and
(DPPH)
obtained for the isolated BA were in accordance with published data. Antioxidant activity
13
C NMR spectroscopic technique.
13
C NMR data
(IC50) of tested materials ranged from 18 to 148 mg/ml, with MeTp having the highest value relative to ascorbic acid, the reference standard. The radical scavenging activities of MeTp and that of BA plus ascorbic acid mixture were greater than that of ascorbic acid alone, indicating synergism between ascorbic acid and betulinic acid. Conclusion: The findings established the antioxidant property of methanolic root extract of T. potatoria. The combination of betulinic acid and ascorbic acid could be useful for the development of new antioxidant drug. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Tetracera potatoria Afzel. ex G. Don, is known as water tree in Sierra-Leone.1 It is found in wooded areas of Senegal, southern part of Nigeria, Central and Eastern Africa.2 It is used for the treatment of backache, diabetes and as an anti-scorbutic.
The leaves of the plant boiled in its own sap are used for the treatment of gastrointestinal sores.1 Its sap is used for toothache and cough.3 It is used in the treatment of jaundice and haemorrhoids among the Baka Pygmies of Cameroon and also used in the traditional treatment of inflammatory, skin infection and ulcer.4,5
* Corresponding author. Tel.: þ234 8030821561. E-mail addresses: [email protected], [email protected] (J.K. Adesanwo). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.09.003
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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j o u r n a l o f p h a r m a c y r e s e a r c h x x x ( 2 0 1 3 ) 1 e5
The presence of alkaloids, tannins, saponins, phlobatannins, terpenoids and flavonoids in the leaves of T. potatoria has been reported.6 T. potatoria root has also been found to contain phytochemicals such as tannins, flavonoids, phlobatannins and cardiac glycosides.7 Betulinic acid, 3b-hydroxy-lup-20(29)-en-28-oic acid, a C-28 carboxylic acid derivative of the ubiquitous triterpene betulin, is a member of the class of the lupane-type pentacyclic triterpenes.
2.2.2.
Test for flavonoids
5 ml dilute ammonia was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1 ml) was added. A yellow colouration that disappears on standing indicates the presence of flavonoids.
2.2.3.
Test for tannins
About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black colouration.
2.2.4.
Test for saponins
To 0.5 g of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.
2.2.5.
It was isolated at the beginning of the 20th century and originally called gratiolone.8 However unlike betulin, the oxidized derivative betulinic acid possesses a number of intriguing pharmacological effects including: antiinflammatory, anticancer and anti-HIV.5,9,10
2.
Methodology
2.1. Collection, processing and extraction of plant material T. potatoria root was collected from Ilesa, Osun state, Nigeria and authenticated by Mr. G. Ibhanesebhor, plant taxonomist, Herbarium, Obafemi Awolowo University, Ile-Ife, Nigeria. Voucher specimen (IFE Herbarium 16419) was deposited in the herbarium. The plant material was air-dried, pulverised and extracted by soaking 1.2 kg sample in aspirator bottles containing distilled methanol at room temperature (25 C) for 48 h. The extract was filtered and solvent was completely removed by vacuum evaporator at 50 C to give viscous mass (18.55 g, 1.5% yield), which was stored inside a dessicator for further usage.
2.2.
Phytochemical screening
Phytochemical screenings of MeTp were performed using standard procedures.11e13
2.2.1.
Test for anthraquinones
0.5 g of the extract was boiled with 10 ml of sulphuric acid (H2SO4) and filtered hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipetted into another test tube and 1 ml of dilute ammonia solution was added. The presence of pink colour in the aqueous layer indicated the presence of anthraquinones.
Test for alkaloids
0.5 g of extract was dissolved in 10 ml alcohol, acidified and boiled and then filtered. To 5 ml of the filtrate was added 2 ml of dilute ammonia. 5 ml of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 ml of acetic acid. This was divided into two portions. Mayer’s reagent was added to one portion and Draggendorff’s reagent to the other. The formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent) was regarded as positive for the presence of alkaloids.
2.3.
Isolation and purification of BA
MeTp (15 g) was fractionated using Accelerated Gradient Chromatography (AGC) to facilitate isolation of BA, according to our earlier report.5 Gradient elution was effected with solvent combination of n-hexane (100%) and a sequential increase in polarity using mixtures of n-hexane/ethyl acetate and ethyl acetate/methanol. A total of 111 fractions (20 ml each) were collected and analysed by TLC using appropriate solvent systems. Fractions with similar TLC profiles were pooled together and concentrated to dryness in vacuo using rotary evaporator. Ten different combined fractions coded as Tp1 (1e9), Tp2 (14e21), Tp3 (24e32), Tp4 (37e52), Tp5 (55e65), Tp6 (66e74), Tp7 (75e85), Tp8 (83e86), Tp9 (93e101) and Tp10 (102e111) were obtained. Fractions Tp2 and Tp3 eluted with 8:2 and 7:3 n-hexane:ethyl acetate, were identical, combined and recrystallized in methanol. This afforded a white crystalline compound A (0.31 g), which was not UV active but showed one spot on TLC plate, under iodine vapour (Rf 0.63 in n-hexane/ethyl acetate 3:2; mpt. 290e293 C). 1H NMR (400 mHz), CDCl3 (ppm): 4.7 (1Hs, H-30); 4.9 (1Hs, H-30); 3.0 (1Hdt, 4, 11 Hz, H-19); 1.7 (3Hs, H-29). 13C NMR is contained in Table 2 below. Other fractions were kept for future analysis.
2.4.
Spectroscopic analysis
The structural elucidation of compound A was carried out using proton, carbon-13, heteronuclear NMR experiments and comparison with literature data. The 1H NMR experiments
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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Table 1 e Phytochemical screening results. Plant extracts
Alkaloids
Tannins
Saponins
Cardiac glycosides
Anthraquinones
Flavonoids
þ
þ
þ
þ
þ
MeTp
Key: þ ¼ Present, ¼ Absent.
were performed on a Bruker Avance 400 MHz spectrometer. The 13C NMR spectra were also recorded on the same instrument at 100 MHz at the University of Winnipeg, Manitoba, Canada. The chemical shift values were reported in ppm relative to TMS as internal standard. Melting points were determined on Gallenkamp electrothermal melting point apparatus.
2.5.
The antioxidant activities of MeTp, isolated BA, and ascorbic acid combined with BA were determined using 1,1-diphenyl2-picrylhydrazyl radical (DPPH) free radical scavenging assay by the method of Brand-Williams.14 The DPPH solution was prepared in distilled ethanol. Ethanolic solutions of samples were prepared (0.18 mg/ml) and diluted serially to achieve concentrations of 0.14, 0.1, 0.08, 0.06, 0.04, 0.02, 0.016, 0.012, and 0.008 mg/ml. 2 ml of freshly prepared ethanolic solution of DPPH was mixed with 2 ml of the sample. The mixture was incubated for 30 min and the absorbance measured against a blank at the end of 30 min at a wavelength of 517 nm using a Shimadzu UV/Vis 1601 apparatus. Inhibition of DPPH free radical in (%), was calculated as follows:
Table 2 e 13C NMR spectra of betulinic acid.
1 3 5 7 9 10 13 14 16 17 18 19 20 21 23 25 26 27 28 29 30
80.9
46.9 150.4
181.6 19.3 109.7
Results and discussion
3.1.
Extraction
3.2.
dC (ppm)15
dC (ppm) observed
39.5 78.1 56.0 34.9 51.0 37.6 38.7 42.9 32.9 56.6 49.8 47.8 151.3 30.3 28.7 16.4 16.3 14.9 178.8 19.5 109.9
39.1 77.2 55.3 34.4 50.3 37.2 38.7 42.4 32.1 55.8 48.9 47.0 150.8 30.5 28.5 16.4 16.3 14.8 177.7 19.4 110.1
Qualitative phytochemical screening
The qualitative chemical tests of the methanol extract revealed the presence of alkaloid, saponin, flavonoid, and tannin (Table 1). Anthraquinone was absent.
3.3.
Structural elucidation of the solid
1 H, 13C, APT, and DEPT NMR data were acquired. The data obtained were in agreement with those reported in literature for betulinic acid (Table 2).
3.4.
1 Asample 100 Inhibition ð%Þ ¼ Ablank
dC (ppm)5
3.
The extraction of the root of T. potatoria (1200 g) with cold methanol afforded 18.55 g crude extract (1.5% yield).
Antioxidant activity
Position
where; Ablank is the absorbance of DPPH and Asample is the absorbance of test sample.
Antioxidant activity
Model of scavenging the stable DPPH radical is a widely used method to evaluate the free radical scavenging ability of various samples.16 The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activities of T. potatoria are given in Table 3. The activity was dose dependent. DPPH antioxidant assay is based on the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolourize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for a visible deep purple colour. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. The radical scavenging activity was expressed in terms of the amount of antioxidant necessary to decrease the initial DPPH absorbance by 50% (IC50). The IC50 value for each sample was determined graphically by plotting the percentage
Table 3 e The antioxidant activity (IC50) of MeTp, isolated BA, ascorbic acid and ascorbic acid plus BA. Species MeTp Betulinic acid Ascorbic acid Ascorbic acid þ Betulinic acid
IC50 (mg/ml) 0.018 0.141 0.037 0.023
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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j o u r n a l o f p h a r m a c y r e s e a r c h x x x ( 2 0 1 3 ) 1 e5
H2C
OH
OH HC O
(DPPH)
+
(DPPH)
OH
H2C
OH HC O
O
H HO
H2C H
0H
+
O
OH
OH HC O
(DPPH)
H
O
H 0H
HO
O
0
Fig. 1 e Oxidation of ascorbic acid to the dehydro form by DPPH radical.
disappearance of DPPH as a function of the sample concentration. The lower the IC50 value, the higher the potential antioxidant activity. IC50 values obtained ranged from 0.018 to 0.148 mg/ml (Table 3). MeTp demonstrated the strongest antioxidant activity (0.018 mg/ml), than ascorbic acid (0.037 mg/ml) and BA (0.141 mg/ml). The mixture of ascorbic acid and betulinic acid also demonstrated stronger activity (0.023 mg/ml) than the reference drug. The antioxidant activity of MeTp, BA and BA plus ascorbic acid mixture decreased in the order: MeTp > BA þ ascorbic acid > ascorbic acid > BA. Generally, an increase in the number of hydroxyl groups (eOH) or other H-donating groups (eNH; eSH) in the molecular structure the higher is the antioxidant activity.17 Plant polyphenols, a diverse group of phenolic compounds (flavanols, flavonols, anthocyanins, phenolic acids, etc.) possess an ideal structural chemistry for free radical scavenging activity. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors from the ability of the polyphenol derived radical to stabilize and delocalize the unpaired electron.18
category has found wide-spread application in the stabilization of hydrocarbon polymers, viz, combinations of chainbreaking antioxidants and preventive antioxidants of various types. In the case of a combination of two different chainbreaking antioxidants (homosynergism) that function by donation of hydrogen to a DPPH radical, the most likely mechanism of synergism would involve transfer of hydrogen from one antioxidant to the radical formed in the reaction of the other antioxidant with a DPPH radical. Typical examples are combinations of hindered phenols with other phenols,20 ascorbic acid,23 dialkylphosphonates24 and aromatic amines.25 In all these cases it is believed that the stronger antioxidant is regenerated from its radical by the less powerful antioxidant, serving as a reservoir of hydrogen for regeneration of the more effective chainbreaking antioxidant. It was also shown that the concentration of the more effective antioxidant remains constant during the oxidation until complete consumption of the weak antioxidant occurred.
3.5.
3.6. Proposed mechanism of synergism between ascorbic acid and BA
Synergism of antioxidants
Synergism of antioxidants is the cooperative effect of antioxidants or an antioxidant with other compounds to produce enhanced activity than the sum of activities of the individual component when used separately.19 There are two mechanistically distinct types of synergism.20e22 Homosynergism, involves two compounds operating by the same mechanism and heterosynergism, arising from the cooperative effect of antioxidants acting by different mechanisms. The latter
HOH2C OH HC OH HC
O
O
O
OH
OH +
O HO
In the combination of ascorbic acid and BA, it is believed that the stronger antioxidant, ascorbic acid, donates a proton to the DPPH radical (Fig. 1), and it is regenerated from its radical by the less powerful antioxidant, BA, serving as a reservoir of hydrogen for regeneration of the more effective chainbreaking antioxidant. The BA radical thus formed is resonance stabilized as shown in Fig. 2.
HOH2C OH HC OH HC
HO
O
O
OH
O
+
O HO
O O HO
Fig. 2 e Reduction of oxidized ascorbic acid by betulinic acid. Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
j o u r n a l o f p h a r m a c y r e s e a r c h x x x ( 2 0 1 3 ) 1 e5
4.
Conclusion
The poor antioxidant activity of betulinic acid may be explained as being due to lack of phenolic group in its structure. Most plant antioxidants generally have phenolic moiety, which can easily donate electrons to reactive radicals because of the resonance stability of phenoxy radical and thus retard radical chain reactions. Crude plant extracts often have greater in-vitro and/or invivo antioxidant activity than isolated constituents at an equivalent dose because of positive interactions (synergism) between components of whole plant extracts, which may explain the high antioxidant activity of T. potatoria methanolic root extract, which contains flavonoid and tannin. Synergism between ascorbic acid and betulinic acid could be explained through chain-breaking electron transfer to DPPH by ascorbic acid and regeneration of ascorbic acid through proton transfer from betulinic acid resulting in a resonance stabilized betulinic acid radical.
Conflicts of interest All authors have none to declare.
Acknowledgement We acknowledge Obafemi Awolowo University for research grant to J. K. Adesanwo.
references
1. Burkill HM. The Useful Plants of West Tropical Africa. 2nd ed. vol. 1. Kew: Royal Botanic Gardens; 1985:650e652. 2. Dalzel JM. The Useful Plants of West Tropical Africa. London: Crown Agent Publication; 1937:495e515. 3. Oliver B. Medicinal Plants in Nigeria. Nigeria: Nigerian College of Arts, Science and Technology; 1960:1e138. 4. Betti JL. An ethnobotanical study of medicinal plants among the Baka pygmies in the Dja biosphere reserve: Cameroon. Afr Study Monogr. 2004;25(1):1e27. 5. Adesanwo JK, Ekundayo O, Oluwole FS, Olajide OA, Van Den Berge AJ, Findlay JA. The effect of Tetracera potatoria Afzel and its constituentdbetulinic acid on gastric acid secretion and experimentally-induced gastric ulceration. Niger J Physiol Sci. 2003;22:21e25.
5
6. Omotayo FO, Borokini TI. Comparative phytochemical and ethnomedicinal survey of selected medicinal plants in Nigeria. Sci Res Essays. 2012;7(9):989e999. 7. Oyebanji OB, Saba AB. Phytochemistry and in vitro antioxidant activities of Stellaria media, Cajanus cajan and Tetracera potatoria methanolic extracts. J Med Plants Res. 2011;5(30):6622e6627. 8. Simonsen JL, Ross WCJ. The Terpenes. vol. 4. University Press Cambridge; 1957:289. 9. Connolly JD, Hill RA. Triterpenoids. Nat Prod Rep. 2000;17:463e482. 10. Krasutsky PA, Carlson RM, Nesterenko VV, Kolomitsyn IV, Edwardson CF. Birch Bark Processing and Isolation of Natural Products from Birch Bark. 2002. U.S. Patent 6,392,070. 11. Sofowora A. Screening Plants for Bioactive Agents: Medicinal Plants and Traditional Medicine in Africa. 2nd ed. Sunshine House, Ibadan, Nigeria: Spectrum Books Ltd.; 1993:134e156. 12. Harborne JB. Phytochemistry. London: Academic Press; 1993:89e131. 13. Trease GE, Evans WC. Pharmacognosy. 15th ed. London: Saunders Publishers; 2002:42e44. 14. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LebensmittelWissenschaft, und-Technologie. 1995;28:25e30. 15. Peng C, Bodenhausen G, Qiu S, et al. Computer-assisted structure elucidation: application of CISOCSES to the resonance assignment and structure generation of betulinic acid. Magn Reson Chem. 1998;36:267e278. 16. Lee SE, Hwang HJ, Ha JS, Jeong HS, Kim JH. Screening of medicinal plant extracts for antioxidant activity. Life Sci. 2003;73:167e179. 17. Son S, Lewis BA. Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: structure activity relationship. J Agric Food Chem. 2002;50:468e472. 18. Chanda S, Dave R. In-vitro models for antioxidant activity evaluation and some medicinal plants possessing antioxidant properties: an overview. Afr J Microbiol Res. 2009;3(13):981e996. 19. Nawar WW. In: Fennema O, ed. Food Chemistry. 2nd ed. New York: Marcel Dekker, Inc.; 1986:139. 20. Emanuel NM, Denisov ET, Maizus ZK. Liquid-phase Oxidation of Hydrocarbons. vol. 158. New York: Plenum; 1967:248e249. No. 3798. 21. De Jonge CRHI. Stabilization and degradation of polymers. Allara DL, Hawkins WL, eds. Adv Chem Ser. 1978;169:399e425. 22. Jirackova L, Pospisil J. Angew Makromol Chem. 1979;82:197. Cited in: De Jonge CRHI. Synergism of antioxidants. Pure Appl Chem. 1983;55(10):1637e1650. 23. Olcott HS, Mattill HA. 1941. Cited in: De Jonge CRHI. Synergism of antioxidants. Pure Appl Chem. 1983;55(10):1637e1650. 24. Knapp GC, Orloff H. American Chemical Society Publications. 1960;5(11). Cited in: De Jonge CRHI. Synergism of antioxidants. Pure Appl Chem. 1983;55(10):1637e1650. 25. Karpukhina GV, Maizus ZK, Meskina MYa, Emanuel NM. Zhurnal Fizicheskoi Khimii. 1970;44:1377.
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria Julius K. Adesanwo*, Oluwatoyin O. Makinde, Craig A. Obafemi Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
article info
abstract
Article history:
Aim: This study was designed to assess the antioxidant property of crude methanolic
Received 29 July 2013
extract of Tetracera potatoria roots (MeTp); a bioactive constituent isolated from it, betulinic
Accepted 2 September 2013
acid (BA) and a combination of BA with ascorbic acid.
Available online xxx
Methods: The crude extract was obtained by cold extraction. Phytochemical screening of the crude extract was carried out using standard methods. Isolation of betulinic acid (BA) was
Keywords:
effected by Accelerated Gradient Chromatography (AGC) fractionation of MeTp. Structural
Tetracera potatoria
elucidation of the isolated BA was done with 1H and
Antioxidant activity
MeTp, isolated BA and BA plus ascorbic acid mixture (1:1) were evaluated for their anti-
Ascorbic acid
oxidant activity by 1,1-diphenyl-2-picrylhydrazyl, (DPPH) method.
Betulinic acid
Results: The phytochemical analysis showed the presence of alkaloids, flavonoids, cardiac
1,1-Diphenyl-2-picrylhydrazyl
glycosides, saponins and tannins. Anthraquinones were absent. The 1H and
(DPPH)
obtained for the isolated BA were in accordance with published data. Antioxidant activity
13
C NMR spectroscopic technique.
13
C NMR data
(IC50) of tested materials ranged from 18 to 148 mg/ml, with MeTp having the highest value relative to ascorbic acid, the reference standard. The radical scavenging activities of MeTp and that of BA plus ascorbic acid mixture were greater than that of ascorbic acid alone, indicating synergism between ascorbic acid and betulinic acid. Conclusion: The findings established the antioxidant property of methanolic root extract of T. potatoria. The combination of betulinic acid and ascorbic acid could be useful for the development of new antioxidant drug. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Tetracera potatoria Afzel. ex G. Don, is known as water tree in Sierra-Leone.1 It is found in wooded areas of Senegal, southern part of Nigeria, Central and Eastern Africa.2 It is used for the treatment of backache, diabetes and as an anti-scorbutic.
The leaves of the plant boiled in its own sap are used for the treatment of gastrointestinal sores.1 Its sap is used for toothache and cough.3 It is used in the treatment of jaundice and haemorrhoids among the Baka Pygmies of Cameroon and also used in the traditional treatment of inflammatory, skin infection and ulcer.4,5
* Corresponding author. Tel.: þ234 8030821561. E-mail addresses: [email protected], [email protected] (J.K. Adesanwo). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.09.003
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
2
j o u r n a l o f p h a r m a c y r e s e a r c h x x x ( 2 0 1 3 ) 1 e5
The presence of alkaloids, tannins, saponins, phlobatannins, terpenoids and flavonoids in the leaves of T. potatoria has been reported.6 T. potatoria root has also been found to contain phytochemicals such as tannins, flavonoids, phlobatannins and cardiac glycosides.7 Betulinic acid, 3b-hydroxy-lup-20(29)-en-28-oic acid, a C-28 carboxylic acid derivative of the ubiquitous triterpene betulin, is a member of the class of the lupane-type pentacyclic triterpenes.
2.2.2.
Test for flavonoids
5 ml dilute ammonia was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1 ml) was added. A yellow colouration that disappears on standing indicates the presence of flavonoids.
2.2.3.
Test for tannins
About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black colouration.
2.2.4.
Test for saponins
To 0.5 g of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.
2.2.5.
It was isolated at the beginning of the 20th century and originally called gratiolone.8 However unlike betulin, the oxidized derivative betulinic acid possesses a number of intriguing pharmacological effects including: antiinflammatory, anticancer and anti-HIV.5,9,10
2.
Methodology
2.1. Collection, processing and extraction of plant material T. potatoria root was collected from Ilesa, Osun state, Nigeria and authenticated by Mr. G. Ibhanesebhor, plant taxonomist, Herbarium, Obafemi Awolowo University, Ile-Ife, Nigeria. Voucher specimen (IFE Herbarium 16419) was deposited in the herbarium. The plant material was air-dried, pulverised and extracted by soaking 1.2 kg sample in aspirator bottles containing distilled methanol at room temperature (25 C) for 48 h. The extract was filtered and solvent was completely removed by vacuum evaporator at 50 C to give viscous mass (18.55 g, 1.5% yield), which was stored inside a dessicator for further usage.
2.2.
Phytochemical screening
Phytochemical screenings of MeTp were performed using standard procedures.11e13
2.2.1.
Test for anthraquinones
0.5 g of the extract was boiled with 10 ml of sulphuric acid (H2SO4) and filtered hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipetted into another test tube and 1 ml of dilute ammonia solution was added. The presence of pink colour in the aqueous layer indicated the presence of anthraquinones.
Test for alkaloids
0.5 g of extract was dissolved in 10 ml alcohol, acidified and boiled and then filtered. To 5 ml of the filtrate was added 2 ml of dilute ammonia. 5 ml of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 ml of acetic acid. This was divided into two portions. Mayer’s reagent was added to one portion and Draggendorff’s reagent to the other. The formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent) was regarded as positive for the presence of alkaloids.
2.3.
Isolation and purification of BA
MeTp (15 g) was fractionated using Accelerated Gradient Chromatography (AGC) to facilitate isolation of BA, according to our earlier report.5 Gradient elution was effected with solvent combination of n-hexane (100%) and a sequential increase in polarity using mixtures of n-hexane/ethyl acetate and ethyl acetate/methanol. A total of 111 fractions (20 ml each) were collected and analysed by TLC using appropriate solvent systems. Fractions with similar TLC profiles were pooled together and concentrated to dryness in vacuo using rotary evaporator. Ten different combined fractions coded as Tp1 (1e9), Tp2 (14e21), Tp3 (24e32), Tp4 (37e52), Tp5 (55e65), Tp6 (66e74), Tp7 (75e85), Tp8 (83e86), Tp9 (93e101) and Tp10 (102e111) were obtained. Fractions Tp2 and Tp3 eluted with 8:2 and 7:3 n-hexane:ethyl acetate, were identical, combined and recrystallized in methanol. This afforded a white crystalline compound A (0.31 g), which was not UV active but showed one spot on TLC plate, under iodine vapour (Rf 0.63 in n-hexane/ethyl acetate 3:2; mpt. 290e293 C). 1H NMR (400 mHz), CDCl3 (ppm): 4.7 (1Hs, H-30); 4.9 (1Hs, H-30); 3.0 (1Hdt, 4, 11 Hz, H-19); 1.7 (3Hs, H-29). 13C NMR is contained in Table 2 below. Other fractions were kept for future analysis.
2.4.
Spectroscopic analysis
The structural elucidation of compound A was carried out using proton, carbon-13, heteronuclear NMR experiments and comparison with literature data. The 1H NMR experiments
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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Table 1 e Phytochemical screening results. Plant extracts
Alkaloids
Tannins
Saponins
Cardiac glycosides
Anthraquinones
Flavonoids
þ
þ
þ
þ
þ
MeTp
Key: þ ¼ Present, ¼ Absent.
were performed on a Bruker Avance 400 MHz spectrometer. The 13C NMR spectra were also recorded on the same instrument at 100 MHz at the University of Winnipeg, Manitoba, Canada. The chemical shift values were reported in ppm relative to TMS as internal standard. Melting points were determined on Gallenkamp electrothermal melting point apparatus.
2.5.
The antioxidant activities of MeTp, isolated BA, and ascorbic acid combined with BA were determined using 1,1-diphenyl2-picrylhydrazyl radical (DPPH) free radical scavenging assay by the method of Brand-Williams.14 The DPPH solution was prepared in distilled ethanol. Ethanolic solutions of samples were prepared (0.18 mg/ml) and diluted serially to achieve concentrations of 0.14, 0.1, 0.08, 0.06, 0.04, 0.02, 0.016, 0.012, and 0.008 mg/ml. 2 ml of freshly prepared ethanolic solution of DPPH was mixed with 2 ml of the sample. The mixture was incubated for 30 min and the absorbance measured against a blank at the end of 30 min at a wavelength of 517 nm using a Shimadzu UV/Vis 1601 apparatus. Inhibition of DPPH free radical in (%), was calculated as follows:
Table 2 e 13C NMR spectra of betulinic acid.
1 3 5 7 9 10 13 14 16 17 18 19 20 21 23 25 26 27 28 29 30
80.9
46.9 150.4
181.6 19.3 109.7
Results and discussion
3.1.
Extraction
3.2.
dC (ppm)15
dC (ppm) observed
39.5 78.1 56.0 34.9 51.0 37.6 38.7 42.9 32.9 56.6 49.8 47.8 151.3 30.3 28.7 16.4 16.3 14.9 178.8 19.5 109.9
39.1 77.2 55.3 34.4 50.3 37.2 38.7 42.4 32.1 55.8 48.9 47.0 150.8 30.5 28.5 16.4 16.3 14.8 177.7 19.4 110.1
Qualitative phytochemical screening
The qualitative chemical tests of the methanol extract revealed the presence of alkaloid, saponin, flavonoid, and tannin (Table 1). Anthraquinone was absent.
3.3.
Structural elucidation of the solid
1 H, 13C, APT, and DEPT NMR data were acquired. The data obtained were in agreement with those reported in literature for betulinic acid (Table 2).
3.4.
1 Asample 100 Inhibition ð%Þ ¼ Ablank
dC (ppm)5
3.
The extraction of the root of T. potatoria (1200 g) with cold methanol afforded 18.55 g crude extract (1.5% yield).
Antioxidant activity
Position
where; Ablank is the absorbance of DPPH and Asample is the absorbance of test sample.
Antioxidant activity
Model of scavenging the stable DPPH radical is a widely used method to evaluate the free radical scavenging ability of various samples.16 The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activities of T. potatoria are given in Table 3. The activity was dose dependent. DPPH antioxidant assay is based on the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolourize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for a visible deep purple colour. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. The radical scavenging activity was expressed in terms of the amount of antioxidant necessary to decrease the initial DPPH absorbance by 50% (IC50). The IC50 value for each sample was determined graphically by plotting the percentage
Table 3 e The antioxidant activity (IC50) of MeTp, isolated BA, ascorbic acid and ascorbic acid plus BA. Species MeTp Betulinic acid Ascorbic acid Ascorbic acid þ Betulinic acid
IC50 (mg/ml) 0.018 0.141 0.037 0.023
Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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H2C
OH
OH HC O
(DPPH)
+
(DPPH)
OH
H2C
OH HC O
O
H HO
H2C H
0H
+
O
OH
OH HC O
(DPPH)
H
O
H 0H
HO
O
0
Fig. 1 e Oxidation of ascorbic acid to the dehydro form by DPPH radical.
disappearance of DPPH as a function of the sample concentration. The lower the IC50 value, the higher the potential antioxidant activity. IC50 values obtained ranged from 0.018 to 0.148 mg/ml (Table 3). MeTp demonstrated the strongest antioxidant activity (0.018 mg/ml), than ascorbic acid (0.037 mg/ml) and BA (0.141 mg/ml). The mixture of ascorbic acid and betulinic acid also demonstrated stronger activity (0.023 mg/ml) than the reference drug. The antioxidant activity of MeTp, BA and BA plus ascorbic acid mixture decreased in the order: MeTp > BA þ ascorbic acid > ascorbic acid > BA. Generally, an increase in the number of hydroxyl groups (eOH) or other H-donating groups (eNH; eSH) in the molecular structure the higher is the antioxidant activity.17 Plant polyphenols, a diverse group of phenolic compounds (flavanols, flavonols, anthocyanins, phenolic acids, etc.) possess an ideal structural chemistry for free radical scavenging activity. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors from the ability of the polyphenol derived radical to stabilize and delocalize the unpaired electron.18
category has found wide-spread application in the stabilization of hydrocarbon polymers, viz, combinations of chainbreaking antioxidants and preventive antioxidants of various types. In the case of a combination of two different chainbreaking antioxidants (homosynergism) that function by donation of hydrogen to a DPPH radical, the most likely mechanism of synergism would involve transfer of hydrogen from one antioxidant to the radical formed in the reaction of the other antioxidant with a DPPH radical. Typical examples are combinations of hindered phenols with other phenols,20 ascorbic acid,23 dialkylphosphonates24 and aromatic amines.25 In all these cases it is believed that the stronger antioxidant is regenerated from its radical by the less powerful antioxidant, serving as a reservoir of hydrogen for regeneration of the more effective chainbreaking antioxidant. It was also shown that the concentration of the more effective antioxidant remains constant during the oxidation until complete consumption of the weak antioxidant occurred.
3.5.
3.6. Proposed mechanism of synergism between ascorbic acid and BA
Synergism of antioxidants
Synergism of antioxidants is the cooperative effect of antioxidants or an antioxidant with other compounds to produce enhanced activity than the sum of activities of the individual component when used separately.19 There are two mechanistically distinct types of synergism.20e22 Homosynergism, involves two compounds operating by the same mechanism and heterosynergism, arising from the cooperative effect of antioxidants acting by different mechanisms. The latter
HOH2C OH HC OH HC
O
O
O
OH
OH +
O HO
In the combination of ascorbic acid and BA, it is believed that the stronger antioxidant, ascorbic acid, donates a proton to the DPPH radical (Fig. 1), and it is regenerated from its radical by the less powerful antioxidant, BA, serving as a reservoir of hydrogen for regeneration of the more effective chainbreaking antioxidant. The BA radical thus formed is resonance stabilized as shown in Fig. 2.
HOH2C OH HC OH HC
HO
O
O
OH
O
+
O HO
O O HO
Fig. 2 e Reduction of oxidized ascorbic acid by betulinic acid. Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003
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4.
Conclusion
The poor antioxidant activity of betulinic acid may be explained as being due to lack of phenolic group in its structure. Most plant antioxidants generally have phenolic moiety, which can easily donate electrons to reactive radicals because of the resonance stability of phenoxy radical and thus retard radical chain reactions. Crude plant extracts often have greater in-vitro and/or invivo antioxidant activity than isolated constituents at an equivalent dose because of positive interactions (synergism) between components of whole plant extracts, which may explain the high antioxidant activity of T. potatoria methanolic root extract, which contains flavonoid and tannin. Synergism between ascorbic acid and betulinic acid could be explained through chain-breaking electron transfer to DPPH by ascorbic acid and regeneration of ascorbic acid through proton transfer from betulinic acid resulting in a resonance stabilized betulinic acid radical.
Conflicts of interest All authors have none to declare.
Acknowledgement We acknowledge Obafemi Awolowo University for research grant to J. K. Adesanwo.
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Please cite this article in press as: Adesanwo JK, et al., Phytochemical analysis and antioxidant activity of methanol extract and betulinic acid isolated from the roots of Tetracera potatoria, Journal of Pharmacy Research (2013), http://dx.doi.org/10.1016/ j.jopr.2013.09.003