In vitro antioxidant and anti-cholinesterase activities of Rhizophora mucronata

Figures & data

Table 1. Acetylcholinesterase inhibitory activities of different solvent extracts of R. mucronata.

		% of inhibition ± S.D.^a
Solvent extract	% of Yield	100 µg/ml	200 µg/ml	300 µg/ml	400 µg/ml	500 µg/ml	IC50 (µg/ml)
Donepezil	1.2	82.91 ± 1.8	83.86 ± 1.8	88.6 ± 1.89	88.60 ± 5.02	94.30 ± 0.00001	5.75 ± 0.04
Petroleum ether	0.19	NI^b	NI	NI	NI	NI	Nil
Hexane	0.28	NI	NI	NI	NI	NI	Nil
Benzene	1.41	NI	NI	NI	NI	NI	Nil
Dichloromethane	0.16	NI	NI	NI	NI	NI	Nil
Chloroform	0.06	39.39 ± 0.13	98.78 ± 0.13	99.09 ± 0.033	99.8 ± 0.000019	99.88 ± 0.000019*	117.86 ± 2.61
Ethyl acetate	0.99	72.23 ± 0.95	78.08 ± 0.47	78.48 ± 2.44	79.98 ± 3.74	81.12 ± 2.67*	69.21 ± 1.98
Acetone	4.77	34.97 ± 3.4	47.53 ± 4.52	56.43 ± 5.42	62.25 ± 1.76	62.32 ± 1.71*	259.56 ± 5.69
Methanol	4	84.30 ± 1.22*	84.55 ± 0.34	87.43 ± 1.14	88.49 ± 0.302	92.73 ± 0.54*	59.31 ± 0.35
Water	1.2	NI	NI	NI	NI	NI	Nil

^aResults were expressed as mean ± SEM (n = 3).

^bNI, no inhibition.

*p < 0.05.

Figure 1. Schematic representation of bioactive guided fractionation of methanolic extract of Rhizophora mucronata.

Table 2. Butyrylcholinesterase inhibitory activities of different solvent extracts of R. mucronata.

	% of inhibition ± S.D.^a
Solvent extract	100 µg/ml	200 µg/ml	300 µg/ml	400 µg/ml	500 µg/ml	IC50 (µg/ml)
Donepezil	90.35 ± 0.53	95.24 ± 0.306	97.52 ± 0.505	98.05 ± 0.11	98.92 ± 0.23	5.53 ± 0.06
Petroleum ether	80.99 ± 2.74	82.59 ± 2.09	90.41 ± 2.82	90.64 ± 0.34	94.54 ± 0.72*	60.21 ± 0.23
Hexane	NI^b	NI	NI	NI	NI	Nil
Benzene	74.45 ± 0.07	86.10 ± 8.9	86.31 ± 6.68	88.13 ± 3.17	90.38 ± 1.12*	67.154 ± 2.9
Dichloromethane	88.07 ± 2.88	87.13 ± 2.0	91.14 ± 0.35	95.16 ± 1.13	95.75 ± 0.70*	57.06 ± 1.20
Chloroform	95.98 ± 0.54	96.93 ± 1.59	97.04 ± 0.20	97.16 ± 0	97.75 ± 0.20*	52.09 ± 0.29
Ethyl acetate	93.50 ± 0.20	93.50 ± 0.20	94.80 ± 0.89	96.45 ± 1.97	97.52 ± 1.84*	53.48 ± 0.40
Acetone	97.27 ± 0.30	97.88 ± 0	98.18 ± 0.30	98.02 ± 0.46	98.48 ± 0.000013	51.64 ± 0.24
Methanol	96.66 ± 1.09	98.07 ± 0.17	98.15 ± 0.52	98.88 ± 0.76	98.98 ± 0.17*	51.72 ± 0.33
Water	77.49 ± 0.76	77.90 ± 8.47	79.40 ± 3.02	79.71 ± 3.78	84.66 ± 1.55*	64.58 ± 1.7

^aResults were expressed as mean ± SEM (n = 3).

^bNI, no inhibition.

*p < 0.05.

Figure 2. (A) Ferric reducing antioxidative power of different solvent extracts of R. mucronata (100–500 µg/ml) in comparison with l-ascorbic acid (100–500 µg/ml). (B) Reducing power of different solvent extracts of R. mucronata (100–500 µg/ml) in comparison with standard l-ascorbic acid, (C) inhibitory effects of R. mucronata extract on DNA nicking caused by hydroxyl radicals, 0.5 µg of pUC18 plasmid DNA to Fenton’s reaction solution in the absence (lane 3) or presence of different solvent fractions of R. mucronata (1 mg/ml) for 30 min at 37 °C [lane 4 (PE), lane 5 (HE), lane 6 (BE), lane 7 (CHL), lane 8 (DM), Lane 9 (EA), lane 10 (Ac), lane 11 (Me), lane 12 (Water). Lanes 1 and 2 show the DNA molecular marker and native plasmid DNA, respectively].

Figure 3. (A) HPTLC chromatogram of methanolic fraction of leaves of R. mucronata (i) p-anisaldehyde sulfuric acid; (ii) plates sprayed with 20% aluminum chloride; (iii) plates sprayed with 10% ferric chloride. (B) HPTLC chromatogram of column fraction F13. Plates sprayed with (a) p-anisaldehyde sulfuric acid; (b) 20% aluminium chloride; (c) 25% antimony trichloride in chloroform.

Table 3. Antioxidative properties of R. mucronata leaves extracted with different solvents.

			% of inhibition
Sample	Total phenolic content/mg of extract (µg gallic acid equivalent/mg)^a	Total flavonoid (μg of rutin equivalent/mg of extract)^a	DPPH	OH^• scavenging assay	NO^• scavenging assay	H2O2 scavenging assay	Metal chelating activity
Std BHT	–	–	96.6 ± 0.07	95.99 ± 0.2	87.77 ± 0.01	98.8 ± 0.06	85.08 ± 0.20
PE	50.65 ± 0.5	–	20.21 ± 0.05	90.65 ± 0.07	54.21 ± 0.02	96.47 ± 0.04	100.0 ± 0.02
HEX	12.36 ± 0.31	–	87 ± 0.02	86.31 ± 0.08	55.84 ± 0.01	26.6 ± 0.02	36.04 ± 0.04
BEN	30.81 ± 0.27	–	67.2 ± 0.05	84.9 ± 0.03	65.48 ± 0.07	76.99 ± 0.02	43.39 ± 0.05
DCM	79.25 ± 0.35	–	89.54 ± 0.0	60.97 ± 0.03	50.63 ± 0.01	90.84 ± 0.09	34.03 ± 0.05
CHL	115.2 ± 0.62	20.9 ± 0.04	74.79 ± 0.01	84.76 ± 0.1	83.69 ± 0.04	41.99 ± 0.08	24.25 ± 0.07
EA	596.75 ± 0.35	42 ± 0.05	60.75 ± 0.07	83.41 ± 0.02	94.86 ± 0.01	95.13 ± 0.04	9.94 ± 0.03
AC	65.68 ± 0.59	42.50 ± 0.03	48.34 ± 0.06	85.54 ± 0.04	84.58 ± 0.19	83.46 ± 0.06	59.41 ± 0.01
MET	598.13 ± 1.85*	48.85 ± 0.70*	91.05 ± 0.07*	95.16 ± 0.02*	89.13 ± 0.06*	96.4 ± 0.04*	90.21 ± 0.02*
H2O	64.99 ± 0.06	41.50 ± 0.04	39.64 ± 0.05	60.45 ± 0.09	57.95 ± 0.01	45.30 ± 0.04	98.02 ± 0.02

^aEach experiment was performed in triplicate and the results are mean ± SD. Values in the table are significantly different (*p < 0.05).

Table 4. Compounds identified by LC-MS analysis in methanolic extract of R. mucronata.

S. no.	Compound name	Molecular mass
1.	Syringic acid	198.18
2.	Catechol	290.28
3.	Beta-amyrin	426.73
4.	Coumaric acid	116.08
5.	Octanal	128.22
6.	Oxal acetic acid	132.08
7.	Myrecene	136.24
8.	Hygrine	141.22
9.	Coumarin	146.15
10.	Coumaryl alcohol	150.18
11.	Caffeoyl alcohol	166.18
12.	Synephrine	167.21
13.	Scopoletin	192.17
14.	Glucuronic acid	194.15
15.	Magnolol	266.34
16.	Linolenic acid	278.44
17.	Rutinose	342.31
18.	Methyl ellagic acid	344.28
19.	Lupeol	426.73
20.	Cysteine sulfoxide	177.22
21.	Xanthotoxin	216.20
22.	Catechin	290.27
23.	3′,4′,5,7-Tetrahydroxy-3,6,8-trimethoxyflavone	376.32

Table 5. Antioxidant and cholinesterase inhibitory activity of fractions and (+)-catechin.

	Antioxidative property			% of inhibition
Sample	DPPH (% inhibition)	FRAP assay (absorbance 532 nm)	Reducing power (absorbance 700 nm)	Acetyl cholinesterase	Butyryl cholinesterase
Fraction 13^a (ethyl acetate:methanol 1:1)	94.42 ± 0.005	0.092 ± 0.012	1.753 ± 0.014	97.83 ± 0.0003	98.58 ± 0.003
Fraction 3 (catechin)^b	89.89 ± 0.005	0.314 ± 0.002	2.01 ± 0.013	93.20 ± 0.0012	92.50 ± 0.0043
Fraction 7^b	80.41 ± 0.001	0.168 ± 0.0004	1.61 ± 0.008	64.78 ± 0.006	88.60 ± 0.001
Fraction 8^b	85.49 ± 0.006	0.131 ± 0.0002	1.11 ± 0.013	75.39 ± 0.001	89.26 ± 0.001
Standard donepezil	–	–	–	99.25 ± 0.00014	94.03 ± 0.00012
Standard BHT/ascorbic acid	84.98 ± 0.005	0.386 ± 0.0015	2.02 ± 0.070	–	–

^aFraction eluted using column chromatography.

^bFraction eluted using RPHPLC.

Figure 4. HPLC chromatogram of (A) fraction 13 eluted by column chromatography and (B) purified active fraction 3.

Figure 5. (A) HPTLC chromatogram of purified compound 1 – (i) ammonia vapors viewed under visible light and (ii) alcoholic aluminum chloride viewed under UV 366 nm. (B) The structure of purified compound 1 – (+)-catechin.

Table 6. Enzyme kinetic study of MERM and bioactive compound.

	AChE			BuChE
Kinetic parameters	Control	MERM	Purified compound 1	Control	MERM	Purified compound 1
Vmax (mM/min/mg of protein)	0.05 ± 0.002	0.043 ± 0.001	0.04 ± 0.0001	0.002 ± 0.0001	0.005 ± 0.0008	0.004 ± 0.0003
Km (mM)	21.49 ± 0.10	9.75 ± 3.30	11.71 ± 6.54	0.97 ± 0.18	10.59 ± 2.01	8.53 ± 0.49
Ki (mM)		0.36 ± 0.001	49.58 ± 0.04	–	127.11 ± 1.07	24.11 ± 2.56
Type of inhibition	–	Competitive	Competitive	–	Mixed type inhibition	Mixed type inhibition