EVALUATING THE ACCUMULATION TREND OF L-DOPA IN DARK-GERMINATED SEEDS AND SUSPENSION CULTURES OF <i>Phaseolus vulgaris</i> L. BY AN EFFICIENT UV-SPECTROPHOTOMETRIC METHOD

Rahmani-Nezhad, Samira; Dianat, Shima; Saeedi, Mina; Tehrani, Maliheh Barazandeh; Ghadiri, Adel; Hadjiakhoondi, Abbas

doi:10.21577/0100-4042.20170193

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Artigo • Quím. Nova 41 (4) • Apr 2018 • https://doi.org/10.21577/0100-4042.20170193 copy

EVALUATING THE ACCUMULATION TREND OF L-DOPA IN DARK-GERMINATED SEEDS AND SUSPENSION CULTURES OF Phaseolus vulgaris L. BY AN EFFICIENT UV-SPECTROPHOTOMETRIC METHOD

Authorship SCIMAGO INSTITUTIONS RANKINGS

Seed germination and plant cell cultures provide an alternative mean for producing secondary metabolites. The present study is an attempt to evaluate the eﬀect of seed dark germination and some elicitors and precursors on the production of L-DOPA in Phaseolus vulgaris L. Callus cultured on Murashige and Skoog medium supplemented with various concentrations of different plant growth regulators. L-DOPA produced was quantiﬁed by UV-spectrophotometric method. In this study, a user-friendly, quick, and economical UV-spectrophotometric method was described to determine L-DOPA content in extracts from 33 biotypes of Phaseolus vulgaris L. The method is based on the nitrosation of L-DOPA to form a yellow solution and then formation of a red solution by adding base which is measurable at 470 nm. According to our statistical studies, this method showed high efficiency and selectivity for quantitative determination of L-DOPA in herbal extracts from dried plant seeds, dark-germinated seeds and callus cultures. L-DOPA content in dark-germinated seeds and suspension cultures increased significantly to approximately several-fold compared to the control. The implication from this study is that elicitor treatment and precursor feeding of Phaseolus vulgaris L. can significantly improve the parkinson’s relevant L-DOPA content.

Keywords:
callus cultures; dark germination; L-DOPA; spectrophotometric determination; Phaseolus vulgaris L

Parameters	Analytical data
Instrumental precision (CV, %) (n=5)	0.16
Repeatability (CV, % ) (n =12)	0.37
Linearity Range (µg mL^-1)	4-36
λmax (nm)	470
ε (L mol ^-1cm ^-1)	7.1944×10³
Sandell sensitivity (µg cm^-2)	0.0273
Slope	0.036
Intercept	0.0067
Standard deviation of Slope (SD)	± 0.002
Standard deviation of intercept (SD)	± 0.028
Correlation co-efficient (r)	0.9978
Accuracy (%)	99.55-100.27
Limit of detection (LOD, µg mL^-1)	1.12
Limit of quantification (LOQ, µg mL^-1)	3.74

Concentration (µg mL^-1)	Intra-dayprecision L-dopafound (µg mL^-1)^a a Mean ± SD, n =3.	RSD %	RE%	Inter-dayprecision L-dopafound (µg mL^-1)^a a Mean ± SD, n =3.	RSD %	RE%
4	4.12 ± 0.24	0.24	0.21	4.27 ± 1.06	1.05	0.27
20	20.67 ± 1.84	0.36	0.21	20.11 ± 1.83	0.36	0.02
28	28.39 ± 1.94	0.27	0.05	28.60 ± 0.60	0.08	0.08

Amount of L-DOPA present (mg)	Added (mg)	Found (mg)^a	Recovery (%)^a
5.06	1.29	6.46 ± 0.09	101.04 ± 1.38
5.09	2.85	8.02 ± 0.06	100.27 ± 0.76
5.05	3.46	8.56 ± 0.08	99.55 ± 0.87
5.07	4.67	9.82 ± 0.07	100.25 ± 0.66

Entry	Sample's code	L-DOPA content (%)^a a Mean ± SD, n=3.
1	Ks10001	0.48 ± 0.02
2	Ks10002	0.39 ± 0.10
3	Ks10003	0.45 ± 0.04
4	Ks10004	0.28 ± 0.21
5	Ks10005	0.25 ± 0.09
6	Ks10007	0.39 ± 0.14
7	Ks10008	0.08 ± 0.08
8	Ks10009	0.36 ± 0.05
9	Ks10010	0.35 ± 0.17
10	Ks10011	0.38 ± 0.07
11	Ks10012	0.32 ± 0.11
12	Ks10013	0.30 ± 0.17
13	Ks10014	0.39 ± 0.22
14	Ks10016	0.40 ± 0.28
15	Ks10017	0.05 ± 0.05
16	Ks10022	0.31 ± 0.04
17	Ks10023	0.41 ± 0.13
18	Ks10024	0.36 ± 0.20
19	Ks10025	0.24 ± 0.36
20	Ks10026	0.40 ± 0.07
21	Ks10027	0.33 ± 0.14
22	Ks10029	0.29 ± 0.23
23	Ks10030	0.28 ± 0.04
24	Ks10031	0.39 ± 0.13
25	Ks10032	0.34 ± 0.19
26	Ks10033	0.30 ± 0.16
27	Ks10034	0.36 ± 0.08
28	Ks10035	0.39 ± 0.03
29	Ks10037	0.36 ± 0.11
30	Ks51102	0.09 ± 0.25
31	Ks51176	0.06 ± 0.29
32	Ks51273	0.05 ± 0.06
33	Ks920176	0.26 ± 0.12

Sample's code (Approximately 300 mg)	Amount of L-DOPA added (mg)	Theoretical content (mg)^a a Theoretical values have been obtained using Table 4 for approximately 300 mg of sample extract, which has been added with 2 mg of L-DOPA.	Amount found (mg)^b a Theoretical values have been obtained using Table 4 for approximately 300 mg of sample extract, which has been added with 2 mg of L-DOPA.	Recovery (%)^a a Theoretical values have been obtained using Table 4 for approximately 300 mg of sample extract, which has been added with 2 mg of L-DOPA.	Repeatability (RSD %)
Ks10001	2.0	3.42	3.43 ± 0.04	100.29 ± 1.27	1.27
Ks10014	2.0	3.16	3.14 ± 0.01	99.57 ± 0.48	0.49
Ks10033	2.0	2.89	2.88 ± 0.02	99.65 ± 0.92	0.92
Ks10025	2.0	2.71	2.70 ± 0.03	99.87 ± 1.40	1.40
Ks51176	2.0	2.17	2.19 ± 0.01	100.92 ± 0.46	0.46

Entry	Sample's code	Based on UV spectrophotometry method			Based on HPLC method
Entry	Sample's code	Recovery (%)	SD^a a Average of 6 determinations.	RSD (%)	Recovery (%)	SD^a a Average of 6 determinations.	RSD (%)	t^b b Theoretical value at 95% confidence level (2.57).
1	Ks10008	99.41	0.57	0.91	99.76	0.30	0.86	1.21
2	Ks10016	99.82	0.23	0.69	99.53	0.27	0.42	0.93
3	Ks10025	99.11	0.13	0.82	99.80	0.24	0.75	0.65
4	Ks10031	99.35	0.47	0.77	99.75	0.31	0.32	1.25
5	Ks10037	99.30	0.50	1.10	100.1	0.37	0.89	1.34
6	Ks51176	99.70	0.10	0.89	99.49	0.29	0.71	0.86
7	Ks920176	99.10	0.33	0.76	99.45	0.23	0.39	0.74
8	M. pruriens	99.54	0.21	0.58	99.36	0.12	0.48	0.49

Entry	Sample's code	Day 0	Day 3	Day 6	Day 9	Day 12
1	Ks10001	0.48	0.60	1.11	1.48	0.96
2	Ks10008	0.08	0.11	0.21	0.28	0.18
3	Ks10017	0.05	0.09	0.24	0.35	0.20
4	Ks10023	0.41	0.53	0.97	1.31	0.89
5	Ks10025	0.24	0.35	1.19	0.84	0.72
6	Ks10032	0.34	0.43	0.78	1.04	0.68
7	Ks51273	0.05	0.09	0.35	0.23	0.11

PGR	Concentration (µM)	FW (g L^-1)	DW (g L^-1)
2,4-D	0.5	21±0.97	1.5±1.24
	1.5	27±1.34	2.3±0.89
	2.5	49±0.69	3.8±0.79
	4.5	41±0.81	3.1±1.35
	11.0	36±1.21	2.7±0.62
	22.5	25±1.36	1.2±1.01
	45.0	19±0.84	0.9±1.44
IAA	0.5	24±1.84	2.2±0.71
	1.5	26±0.54	1.8±0.89
	3.0	32±0.65	2.3±1.37
	5.5	45±1.65	4.1±0.74
	14.0	38±0.94	2.9±1.21
	28.0	35±1.54	2.7±0.81
	57.5	32±1.77	3.5±1.10
BA	0.4	14±1.37	1.1±0.96
	1.5	28±1.09	2.3±0.73
	2.0	44±1.40	3.5±0.90
	4.5	22±0.81	1.9±1.10
	11.0	20±1.06	1.5±1.04
	22.5	18±1.59	1.1±0.64
	44.0	16±1.29	1.2±1.27
2-iP	0.6	17±0.95	0.9±1.33
	1.5	21±0.76	1.6±1.53
	3.00	48±1.00	4.2±1.11
	4.50	26±1.32	2.7±1.22
	12.00	22±0.81	1.6±1.01
	24.50	15±0.79	1.0±0.91
	50.00	11±1.24	0.8±1.59

Day	Concentration of methyl jasmonate in µmol L^-1
Day	Control	50	100	150	200
3	0.95±0.05	1.53±0.82	3.01±0.12	4.32±1.42	9.22±1.02
6	1.31±0.23	2.68±1.32	5.68±0.42	6.31±0.87	10.65±1.45
9	2.01±0.12	4.15±0.65	8.42±0.72	9.26±0.72	14.55±1.67
12	2.26±1.09	3.25±0.21	6.51±0.10	5.55±0.56	11.37±1.25

Day	Concentration of L-tyrosine in mmol L^-1
Day	Control	50	100	150	200
3	0.95±0.05	3.68±0.08	7.65±0.78	9.65±1.34	15.21±1.25
6	1.31±0.23	5.55±0.03	11.37±0.12	17.85±0.09	22.01±1.04
9	2.01±0.12	9.37±0.72	16.37±1.11	21.21±0.41	34.14±1.65
12	2.26±1.09	10.36±1.09	20.35±1.32	32.35±0.65	40.12±1.00

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