1. EFFECT OF DIFFERENT SOLVENT EXTRACTS ON PHENOLIC, FLAVONOID
AND ANTIOXIDANT CONTENT; AND ANTIPROLIFERATIVE ACTIVITY OF CLINACANTHUS NUTANS LEAVES EXTRACT
NOR HASYIMAH HARON1, RAFEDAH ABAS1, ZALEHA MD TOHA1, MOHAMMAD RAZAK HAMDAN2,
NIZUWAN AZMAN1, MOHD RAZIP SAMIAN3, HASNI ARSAD1*
1Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang
2Centre for Drug Research, Universiti Sains Malaysia, Penang
3Laboratory of Biocrystallography and Structural Bioinformatics, Universiti Sains Malaysia, Penang
*Correspondence author:
INTRODUCTION
Clinacanthus nutans (CN) (family Acanthaceae), is a well-known Thailand and Indonesia traditional medicine. In Malaysia, it is commonly known as Sabah snake grass or belalai gajah.
CN leaves have been used as antidiabetic, blood pressure lowering, antilytic and diuretic properties [1][2] besides treating herpes simplex virus [3]. In Malaysia and Singapore, the leaves are frequently taken as drinks for treating or preventing cancer. So far, there is still lack of information as well as its efficacy as an anticancer herb.
OBJECTIVES
Figure 2: Antioxidant activity of different solvent extracts of CN at 2 mg/ml.
Error bars show the variations of three determinations in terms of standard deviation.
To determine the total phenolic, flavonoid and antioxidant content; and antiproliferative activity of different solvent extracts of CN leaves.
IC50 = 4000 ug/mL
IC50 = 1300 ug/mL
IC50 = 200 ug/mL
IC50 = 80 ug/mL
METHODOLOGY
CN leaves
1) Crude water
2) Crude 80% methanol
Partitioned extract
3) Hexane
4) Chloroform
5) Aqueous
LLE
Samples were collected on June 2014 at Pongsu Seribu, Pulau Pinang.
Plant material
Cytotoxicity assay
Extraction
GC-MS
Apoptosis analysis
TPC & TFC
Antioxidant analysis
Statistical analysis
Figure 3: ((a) and (b)) Antiproliferative effect of CN different solvent extracts on HeLa cells at 24 h treatment. (a): Aqueous, 80% MeOh and Water extracts and (b) Hexane and DCM extracts.
1.34%
0.02%
4.01%
11.71%
17.75%
13.82%
10.19%
65.46%
99.21%
Figure 1: Research flow
30.24%
2.96%
0.74%
RESULTS & DISCUSSIONS
Figure 4: DCM extract treatment induces apoptosis in HeLa cells.
Annexin V assays analyzing in HeLa treated with IC20, IC50 and IC80 DCM of 24 h. The sum of the percentages of Annexin V, CF647 and 7-AAD positive cells was calculated. This preliminary results was performed with no replicates.
TPC of CN’s different solvent extracts was in the order of 80% methanol> Aqueous> Water> DCM and Hexane, while TFC was DCM> 80% methanol> Water> Aqueous and Hexane as shown in Table 1.
This result was not parallel with previous study as plant secondary metabolites profile may vary qualitatively and quantitatively even among different batches of the same plant part collected at different time [4].
DPPH and ABTS assay showed the highest antioxidant content for different extracts; crude 80% methanol (21.2 ± 0.5 mg TE/g) and aqueous extract (12.2 ± 0.0 mg TE/g) respectively (Figure 2).
This different result might be due to the different assay that have been used. Antioxidant activity was not detected in water extract while ABTS did. This might be due to DPPH· can only be dissolved in organic media (especially alcoholic) and not in aqueous media, which is an important limitation when interpreting the role of hydrophilic antioxidants [5].
The DCM extract was found to induce cytotoxicity against HeLa cell line with the least IC50 value of 80 µg/mL for 24 h treatment (Figure 3). This extract was then selected for further analysis.
The highest TFC in DCM extract might contributed to its antiproliferative activity. The flavonoids, which contain hydroxyl groups, are found to be responsible for the radical scavenging effect in the plants [6].
Preliminary flow cytometric data showed the progression of apoptosis with the increasing dose of treatment with IC20, IC50 and IC80 DCM for 24 h, where treatment at IC20 had already induced high affinity for Annexin V (Figure 4).
Little references for apoptosis analysis of CN extracts. A study by Fong, 2015 found that cold aqueous leaf extract of CN showed more viable cells of D24 at a lower dose.
Twenty seven known compounds were identified in DCM extract by GC-MS analysis (Figure 5). The major compound was N-(4-Methoxyphenyl)-2-hydroxyimino-acetamide with relative peak area of 4.72%. Previous reports suggested that this compound has antibiotics, antiseptics, chemotherapeutics and antiviral activities [8].
Abundance
min
𝑡𝑅 : 14.44
Figure 5: Phytochemical constituents detected using GC-MS from DCM extract of CN.
Arrow shows the N-(4-Methoxyphenyl)-2-hydroxyimino-acetamide compound.
CONCLUSIONS
The results of our study demonstrated that due to high TPC, 80% methanol extract of CN leaves had high free radical scavenging and reducing activities by DPPH assay.
TFC was the highest in DCM extract and this might be due to high content of flavonoids.
Flow cytometric analysis showed progression of apoptosis with the increasing dose of treatment with IC20, IC50 and IC80 DCM for 24 h.
REFERENCES
P. Wanikiat, A. Panthong, P. Sujayanon, C. Yoosook, A. G. Rossi, and V. Reutrakul, “The anti-inflammatory effects and the inhibition of neutrophil responsiveness by Barleria lupulina andClinacanthus nutans extracts,” Journal of Ethnopharmacology, vol. 116, no. 2, pp. 234–244, 2008.
Yong, Y.K.; Tan, J.J.; Teh, S.S.; Mah, S.H.; Ee, G.C.L.; Chiong, H.S.; Ahmad, Z. Clinacanthus nutans Extracts Are Antioxidant with Antiproliferative Effect on Cultured Human Cancer Cell Lines. Evid. Based Complement. Alternat. Med. 2013, 2013:
T. Vachirayonstien, D. Promkhatkaew, M. Bunjob, A. Chueyprom, P. Chavalittumrong, and P. Sawanpanyalert, “Molecular evaluation of extracellular activity of medicinal herb Clinacanthus nutansagainst herpes simplex virus type-2,” Natural Product Research, vol. 24, no. 3, pp. 236–245, 2010.
Silva, G.L., Lee, I. & Kinghorn, A.D. (1998). Special problems with the extraction of plants. In Cannell, R.J.P. (Ed.), Methods in Biotechnology, Vol 4 Natural Products Isolation (pp ). Totowa, NJ: Humana Press Inc.
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Das NP, Pereira TA (1990). Effects of flavonoids on thermal autooxidation of Palm oil: structure- activity relationship. J. Am. Oil Chem. Soc., 67:
Fong, S 2015, Genetic, phytochemical and bioactivity studies of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae), Doctor of Philosophy (PhD), Applied Science, RMIT University.
Table 1: Total phenol, flavonoid and yields of the extracts from CN leaves
Crude extract
Partitioned extracts
Extraction yield (%)
TPC*
(mg GAE/g extract)
TFC*
(mg QE/g extract)
80% methanol
4.32
21.51 ± 0.30
14.04 ± 0.32
Hexane
0.21
2.32 ± 0.19
4.15 ± 0.00
DCM
0.13
11.65 ± 0.06
28.43 ± 1.49
Aqueous
3.70
20.22 ± 0.34
9.63 ± 0.04
Crude water
19.05 ± 0.19**
13.17 ± 0.29**
ACKNOWLEDGEMENT
*Values are expressed as mean ± standard deviation (n=3).
** Significantly correlated by Spearman's rank correlation analysis (p< 0.05)
This work was supported in part by the FRGS grant (203/CIPPT/ ) from Ministry of Higher Education (MOHE) Government of Malaysia to Hasni A, PhD.