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Research Article
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Biological Activity of Merremia emarginata Crude Extracts in Different Solvents
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A.V. Babu,
R.S.C. Rao,
K.G. Kumar,
B.H. Babu
and
P.V.V. Satyanarayana
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ABSTRACT
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The plant Merremia emarginata (Burm. f.) Hallier f., belongs to Convolvulaceae family. In traditional medicinal system, different parts of M. emarginata have been mentioned to be therapeutically used as deobstruent, diuretic, for cough, headache, neuralgia and rheumatism. In the present study, biological activities of different solvent extracts isolated from M. emarginata were tested. Hexane (IA), ethyl acetate (IB), methanol (IC) and aqueous methanol (25%) (ID) extracts of M. emarginata were examined. Antioxidant properties of the extracts were studied by DPPH (1,1-Diphenyl-2-Picrylhydrozyl) radical scavenging activity method and superoxide radical scavenging activity method. Methanol extract exhibited better antioxidant activity than other extracts with IC50 of 8.59 μg mL-1 in DPPH radical scavenging method. Methanol and hexane extracts exhibited α-amylase inhibitory activity with IC50 of 104.5 and 133.4 μg mL-1, respectively. Ethyl acetate extract showed cytotoxicity with ED50 of 34.29 μg mL-1 in brine shrimp lethality assay. The present study revealed that the extracts IB and IC of M. emarginata were found to be showed promising biological activities. Methanol extract of this plant might be use full for antioxidant and antiobesity activities with minimal toxicity.
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INTRODUCTION
Natural products produced by plants, microorganisms, insects and animals have
been isolated as biologically active pharmacophores (Gordon
and David, 2005; Cragg and Newman, 2007; Wang
et al., 2006). Approximately one-third of the top-selling drugs in
the world are natural products or their derivatives often with ethno pharmacological
background (Kingston, 2009). The advantage of natural
products for random screening is the structural diversity provided by natural
products, which is greater than provided by most available combinatorial approaches
based on heterocyclic compounds (Harvey, 1999). Cox
(1994) suggested that the ethno-directed sampling is most likely to succeed
in identifying drugs used in the treatment of cancer, inflammatory and dermatological
complaints. According to Chatterjee et al. (2006)
tribal healers in most of the countries, the ethnomedical treatment is frequently
used to treat cut wounds, skin infection, swelling, aging, mental illness, cancer,
asthma, diabetes, jaundice, scabies, eczema, venereal diseases, snakebite and
gastric ulcer, provide instructions to local people as how to prepare medicine
from herbs. India possesses rich floristic wealth and diversified genetic resources
of medicinal plants (Arora et al., 2003).
The use of the plant extracts and pure compounds isolated from natural sources
provided the foundation to modern pharmaceutical compounds (Alluri
et al., 2006).
Antioxidants that scavenge reactive oxygen species may be of great value in
preventing the onset and propagation of oxidative stress related diseases such
as autoimmune (Willet, 1994) cardiovascular (Vinson
et al., 1995) and neurovascular diseases (Aggarwal
and Harikumar, 2008). Recently, more attention has been paid to the role
of natural antioxidants, mainly phenolic compounds, which may have higher antioxidant
activities than those of conventional vitamins C, E and β-carotene (Hafidh
et al., 2009). Cytotoxicity screening models like brine shrimp lethality
assay are the preliminary methods for selection of active plant extracts against
cancer (Al-Fatimi et al., 2007). The management
of diabetes by chemical drugs without any side effects is still a challenge
to the medical system. Many efforts have been made to identify new antidiabetic
agents from different sources, especially medicinal plants because of their
effectiveness, fewer side effects and relatively low cost (Bhandari
et al., 2008). The stable blood glucose level is important for diabetic
patients, because it prevents the hyperglycaemia and the complications associated
with diabetes. Carbohydrate hydrolyzing enzyme like α-amylase inhibitors
are one of the essential drugs for managing type II diabetes (Nickavar
et al., 2008).
The plant Merremia emarginata (Burm. f.) Hallier f., belongs to Convolvulaceae
family. In Sanskrit, it is called as Mooshikakarnee (Satyavati
et al., 1987). In India, it is mainly found in Chennai and in some
places of Andhra Pradesh (Pullaiah et al., 2000).
The plant was therapeutically used as deobstruent, diuretic, for cough, headache,
neuralgia and rheumatism (Chatterjee and Prakashi, 1995).
The importance of M. emarginata as a biologically potent plant species
was proposed the study in vitro antioxidant, α-amylase inhibition
and cytotoxicity activities of different solvent extracts of M. emarginata
is reported.
MATERIALS AND METHODS
Plant Material
The plant material (whole plant except flowers) was collected from the Botanical
Garden of Acharya Nagarjuna University, Nagarjuna Nagar and authenticated by
the Department of Botany.
Preparation of Extracts
The plant material was collected and air dried. Finely powdered plant material
(1 kg) was extracted twice with hexane (5 L) and then the spent was continuously
extracted with ethyl acetate, methanol and aqueous methanol (accordingly). The
solvent extracts were collected and dried by using rotary vacuum evaporator.
The dried weights of the crude extracts are as follows hexane (IA):12.3 g, ethyl
acetate (IB):16.65 g, methanol (IC): 30 g and aqueous methanol (ID): 15 g.
Antioxidant Activity
DPPH Radical Scavenging Activity Method
The DPPH (1,1-Diphenyl-2-Picrylhydrozyl) radical scavenging activity was
determined according to the method of Szabo et al.
(2007), the Optical Density (OD) of colored methanolic solution of the DPPH
free radicals was measured at 517 nm. Percent of inhibition was calculated by
comparing absorbance of crude extract with that of control. The radical scavenging
activity of the crude extracts was expressed as the percent of inhibition and
the IC50 values were obtained from the plot drawn concentration (μg)
verses % of inhibition.
Superoxide Radical Scavenging Activity Method
Superoxide radicals were generated in vitro by non enzymatic system
and determined spectrophotometrically (560 nm) by Nitro Blue Tetrazolium (NBT)
photo reduction method (McCord and Fridovich, 1969).
The assay mixture consist of 6.6 mM EDTA containing 3 μg of NaCN, 2 μM
of riboflavin, 50 μM of NBT, crude extract and 67 mM of phosphate buffer
(pH 7.8) in a final volume of 3 mL. The optical density at 560 nm was measured
before and after 15 min illumination. The superoxide radical scavenging activity
of the crude extracts was expressed in IC50 values.
Alpha-Amylase Inhibitory Activity
The α-amylase activity was measured using the dinitrosalicylic acid
(DNS) method developed by Bernfeld (1955), improved by
Jamieson et al. (1969) and adopted for testing
α-amylase inhibitory potential (Da silva et al.,
2004) using 1% soluble starch as substrate. The test substance was pre-incubated
with amylase (100 μL) at room temperature for 20 min prior to the addition
of 100 μL of the substrate solution followed by incubation at 37°C
for 10 min. The reactions were stopped by the addition of 200 μL of DNS
reagent followed by color development by placing the tubes in boiling water
for 5 min and then added 3.6 mL of distilled water. Acarbose was used as positive
control. The absorbance was read at 470 nm and experiments were carried out
in duplicates.
Brine Shrimp Lethality Bioassay
Brine shrimp (Artemia salina) nauplii were hatched using brine shrimp
eggs in a conical shaped vessel (1 L), filled with sterile artificial sea water
of 38 g L-1 of sea salt and adjusted to pH 8.5 using 1 N of NaOH
and kept under constant aeration for 48 h. After hatching, 10 nauplii were drawn
through a pipette and placed in each vial containing 4.5 mL brine solution and
added various concentrations of crude extracts (0-300 μg mL-1)
and the final volume was made up to 5 mL using brine solution and maintained
37°C for 24 h under the light of incandescent lamps (McLaughlin
et al., 1993, 1998). Assays were carried out
in duplicates. The percentage lethality was determined by comparing the mean
surviving larvae of test and control tubes. Podophyllotoxin was used as a positive
control.
RESULTS
The crude extracts IA, IB, IC and ID were studied for antioxidant, α-amylase
inhibitory and cytotoxic activities. The antioxidant activity was measured in
two biological methods namely DPPH and superoxide radical scavenging activity
method. The results of the two methods were incorporated in Table
1. In DPPH method, the IC crude extract showed better anti-oxidant activity
(8.59 μg mL-1) followed by IB (21.5 μg mL-1),
ID (31.2 μg mL-1) and IA (56.2 μg mL-1). In
superoxide radical scavenging activity method, the extract IB exhibited IC50
at 45.1 μg mL-1 followed by IC (54.3 μg mL-1),
IA (87.09 μg mL-1) and ID (89.4 μg mL-1).
Table 1: |
Antioxidant activity of the crude extracts by DPPH and superoxide
radical scavenging methods |
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IA, IB, IC and ID are crude extracts of hexane, ethyl acetate,
methanol and aqueous methanol, respectively |
Table 2: |
Alpha amylase inhibitory activity of the crude extracts |
 |
IA, IB, IC and ID are crude extracts of hexane, ethyl acetate,
methanol and aqueous methanol, respectively |
Table 3: |
Brine shrimp lethality test for the crude extracts |
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IA, IB, IC and ID are crude extracts of hexane, ethyl acetate,
methanol and aqueous methanol, respectively |
The in vitro α-amylase inhibitory activity (Table
2) results revealed that the extract IC showed IC50 at 104.5
μg mL-1 followed by IA. The brine shrimp results were incorporated
in Table 3, the extract IB showed good cytotoxicity against
brine shrimp larvae followed by IA. The ED50 value of the standard
podophyllotoxin is 2.36 μg mL-1.
DISCUSSION
We know that variety of herbs and herbal extracts contain different phytochemicals
with biological activity that can be of valuable therapeutic index. Different
phytochemicals have been found to possess a wide range of activities, which
may help in protection against chronic diseases (Fabricant
and Farnsworth, 2001). Oxygen is easily activated by Ultra Violet (UV) radiation
and heat from the sunlight to produce toxic Reactive Oxygen Species (ROS). These
ROS are highly reactive because they can interact with a number of cellular
molecules and metabolites there by leading to a number of destructive processes
causing cellular damage (Hafidh et al., 2009). Researchers have been
reported that convolvulaceae member like Ipomoea aquatic and I. batatas
L. was showed potent antioxidant properties in their leaves and shoots.
Flavonoids and phenolic compounds widely distributed in plants which have been
reported to exert multiple biological effect including antioxidant , free radical
scavenging abilities, anti-inflammatory, anticarcinogenic etc. (Aher
et al., 2009). The chloroform and ethyl acetate extracts of the aerial
part of Marsilea quadrifolia have been reported profound antibacterial,
cytotoxic and antioxidant effects (Ripa et al., 2009).
The free radical scavenging activity of the 70% aqueous methanol and ethyl acetate
extracts of Annona senegalensis leaves is due to their constituents of
polyphenols (Potchoo et al., 2008). In the present
study, methanol and ethyl acetate extracts from M. emarginata were found
to be showed better antioxidant activities than other extracts.
Alpha-amylase inhibitors are drug-design targets for the development of compounds
for treatment of diabetes, obesity and hyperlipaemia (Franco
et al., 2000). Nickavar and Mosazadeh (2009)
reported that three Morus species extracts have been exhibited in
vitro α-amylase inhibitory activity with IC50 of 12 mg to
18 mg mL-1. Methanol and hexane extracts of M. emarginata
were found to be showed in vitro α-amylase inhibition with IC50
of 104.5 and 133.4 μg mL-1, respectively. These results revealed
that the IC extract has superior α-amylase inhibition activity than other
substrates.
Brine shrimp lethality assay is considered as a useful tool for preliminary
assessment of toxicity and it has been used for the detection plant extract
toxicity (McLauglin et al., 1991). The use of the
brine shrimp assay to screen plant extracts has been used successfully to biomonitor
the isolation of cytotoxic, antimalarial, insecticidal and antifeedent compounds
from plant extracts (Alluri et al., 2006). Moshi
et al. (2009) reported that the extracts from different plants exhibited
toxicity against brine shrimp larvae with LC50 values ranging from
15.35-374 μg mL-1, ethyl acetate and dichloromethane extracts
have been showed more cytotoxicity. In the present study, ethyl acetate extract
from M. emarginata was showed better cytotoxicity effect than other extracts.
Only few reports are available on biological activities of Merremia species.
Austin (2007) reviewed the uses of Merremia dissecta
(Convolvulaceae), the plant has been used for the treatment of inflammations,
itching, snake bites and as intoxicant. The extracts of M. emarginata,
belongs to the same family have not been reported previously for biological
properties.
CONCLUSION In traditional medicinal system, different parts of M. emarginata have been mentioned to be therapeutically used as deobstruent, diuretic, for cough, headache, neuralgia and rheumatism. The present study revealed that the ethyl acetate (IB) and methanol extracts (IC) of M. emarginata were found to be showed promising biological activities. Methanol extract of this plant might be use full for antioxidant and antidiabetic activities with minimal toxicity. ACKNOWLEDGMENT The authors wish to thank Dr. G. Trimurthulu, Vice-President, Laila Impex R and D Centre, Vijayawada, for his help in biological activity studies.
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REFERENCES |
1: Aggarwal, B.B. and K.B. Harikumar, 2009. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int. J. Biochem. Cell Biol., 41: 40-59. CrossRef | Direct Link |
2: Aher, A.N., S.C. Pal, S.K. Yadav, U.K. Patil and S. Bhattacharya, 2009. Antioxidant activity of isolated phytoconstituents from Casuarina equisetifolia frost (Casuarinaceae). J. Plant Sci., 4: 15-20. CrossRef | Direct Link |
3: Al-Fatimi, M., M. Wurster, G. Schroder and U. Lindequist, 2007. Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen. J. Ethnopharmacol., 111: 657-666. CrossRef | Direct Link |
4: Arora, S., K. Kaur and S. Kaur, 2003. Indian medicinal plants as a reservoir of protective phytochemicals. Teratogenesis Carcinogenesis Mutagenesis, 1: 295-300. CrossRef | PubMed |
5: Austin, D.F, 2007. Merremia dissecta, (Convolvulaceae): Condiment, medicine, ornamental and weed: A review. Econ. Bot., 61: 109-120. Direct Link |
6: Bhandari, M.R., N. Jong-Anurakkun, G. Hong and J. Kawabata, 2008. α-Glucosidase and α-amylase inhibitory activities of nepalese medicinal herb. Food Chem., 106: 247-252. CrossRef |
7: Chatterjee, I., A.K. Chakravarty and A. Gomesa, 2006. Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R. Br. J. Ethnopharmacol., 106: 38-43. PubMed | Direct Link |
8: Cox, P.A, 1994. The ethnobotanical approach to drug discovery: strengths and limitations. Ciba Found. Symp., 185: 25-36. PubMed | Direct Link |
9: Cragg, G.M. and D.J. Newman, 2005. Plants as a source of anti-cancer agents. J. Ethnoharmacol., 100: 72-79. CrossRef | PubMed | Direct Link |
10: Newman, D.J. and G.M. Cragg, 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod., 70: 461-477. CrossRef | Direct Link |
11: Da Silva, M.C.M., L.V. Mello, M.V. Coutinho, D.J. Rigden, G. Neshich. and M.F. Grossi-de-Sa et al., 2004. Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha amylase pesq. Agropec.Bras. Brasilia., 39: 201-208. Direct Link |
12: Fabricant, D.S. and N.R. Farnsworth, 2001. The value of plants used in traditional medicine for drug discovery. Environ. Health Perspect., 109: 69-75. PubMed | Direct Link |
13: Hafidh, R.R., A.S. Abdulamir, F.A. Bakar, F. Abas, F. Jahanshiri and Z. Sekawi, 2009. Antioxidant research in asia in the period from 2000-2008. Am. J. Pharmacol. Toxicol., 4: 56-74. CrossRef | Direct Link |
14: Harvey, A.L., 1999. Medicines from nature: Are natural products still relevant to drug discovery? Trends Pharmacol. Sci., 20: 196-198. CrossRef | PubMed |
15: Jamieson, A.D., K.M. Pruitt and C.R.C. Caldwell, 1969. An improved amylase assay. J. Dental Res., 18: 483-483. CrossRef |
16: Kingston, D.G.I., 2009. Tubilin-interactive natural products as anticancer agents. J. Nat. Prod., 72: 507-515. CrossRef | Direct Link |
17: Krishnaraju, A.V., T.V.N. Rao, D. Sundararaju, M. Vanisree, H.S. Tsay and G.V. Subbaraju, 2006. Biological screening of medicinal plants collected from Eastern Ghats of India using Artemia salina (Brine Shrimp Test). Int. J. Applied Sci. Eng., 4: 115-125. Direct Link |
18: McCord, J.M. and I. Fridovich, 1969. Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem., 244: 6049-6055. Direct Link |
19: McLaughlin, J.L., L.L. Rogers and J.E. Anderson, 1998. The use of biological assays to evaluate botanicals. Drug Inform. J., 32: 513-524. CrossRef | Direct Link |
20: Moshi, M.J., E. Innocent, P.J. Masimba, D.F. Otieno and A. Weisheit et al., 2009. Antimicrobial and brine shrimp toxicity of some plants used in traditional medicine in Bukoba District, north-western Tanzania. Tanzania J. Health Resh., 11: 23-28. PubMed | Direct Link |
21: Nickavar, B., L. Abolhasani and H. Izadpanah, 2008. α-Amylase inhibitory activities of six Salvia species., Iran. J. Pharm. Res., 7: 297-303. Direct Link |
22: Nickavar, B. and G. Mosazadeh, 2009. Influence of three Morus, species extracts on α-amylase activity. Iran. J. Pharma. Res., 8: 115-119.
23: Franco, O.L., D.J. Rigden, F.R. Melo, C. Bloch Jr., C.P. Silva and M.F. Grossi de Sa, 2000. Activity of wheat α-amylase inhibitors towards bruchid α-amylases and structural explanation of observed specificities. Eur. J. Biochem., 267: 2166-2173. CrossRef | PubMed | Direct Link |
24: Potchoo, Y., I.P. Guissou, M. Lompo, E. Sakie and B. Yaro, 2008. Antioxidant activity of aqueous methanol and ethyl acetate extract of leaves of Annona senegalensis Pers from togo versus the one originates from Burkina Faso. Int. J. Pharmacol., 4: 120-124. CrossRef | Direct Link |
25: Ripa, F.A.L. Nahar, M.H. and M.M. Islam, 2009. Antibacterial, cytotoxic and antioxidant activity of crude extract of Marsilea quadrifolia. Eur. J. Sci. Res., 33: 123-129. Direct Link |
26: Szabo, M.R., C. Iditoiu, D. Chambre and A.X. Lupea, 2007. Improved DPPH determination for antioxidant activity spectrophotometric assay. Chem. Papers, 61: 214-216. CrossRef |
27: Wang, R., J.S. Yang. L.I. Chen and Y. Zheng, 2006. Anti-HIV-1 activites of extracts from the medicinal plant Rhus Chinensis. J. Ethnopharmacol., 105: 269-273. CrossRef |
28: Vinson, J.A., Y.A. Dabbagh, M.M. Serry and J. Jang, 1995. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food Chem., 43: 2800-2802. Direct Link |
29: Bernfeld, P., 1955. Amylases Alpha and Beta. In: Methods in Enzymology, Colowick, S.B. and N.O. Kaplan (Eds.). Academic Press, New York, pp: 149-153.
30: Satyavati, G.V., A.K. Gupta and T. Neeraj, 1987. Medicinal Plants of India. Vol. 2, India Council of Medical Research, New Delhi.
31: Pullaiah, T., V. Rama Krishna, S. Sandya Rani and P.N. Rao, 2000. Systematic Enumeration: Flora of Guntur District. Regency Publications, New Delhi, ISBN: 81-87498-16-1, pp: 243-244.
32: McLaughlin, J.L., C.J. Chang and D.L. Smith, 1991. Bench Top Bioassay for the Discovery of Bioactive Natural Products: An Update. In: Studies in Natural Product Chemistry, Rahman, A.U. (Ed.). Elseiver, UK., pp: 383-409.
33: McLaughlin, J.L., C.J. Chang and D.L. Smith, 1993. Simple bench-top bioassays (brine shrimp and potato discs) for the discovery of plant antitumor compounds. Am. Chem. Soc. Sympos. Ser., 534: 112-134. CrossRef |
34: Chatterjee, A. and S.C. Prakashi, 1995. The Treatise on Indian Medicinal Plants. Vol. 4, NISC/CSIR, Govt. of India, New Delhi, ISBN: 81-7236-115-7, pp: 148-149.
35: Willett, W.C., 1994. Diet and health: What should we eat? Science, 264: 532-537. CrossRef | Direct Link |
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