Optimization of the extraction conditions and quantification by RP-LC analysis of three alkaloids in Zanthoxylum nitidum roots

Figures & data

Table 1. The specific information of the 20 samples of Z. nitidum roots.

Samples no.	Producing area^a	Collecting time	Specimen no.
1	Daxin County	August 2005	20051001
2	Qinzhou City	July 2006	20061001
3	Ningming County	August 2006	20061002
4	Tiandong County	September 2006	20061003
5	Wuming County	June 2007	20071001
6	Tiandeng County	July 2007	20071002
7	Lingshan County	September 2007	20071003
8	Nanning City	October 2007	20071004
9	Baise City	October2007	20071005
10	Bama County	October 2007	20071006
11	Napo County	October2007	20071007
12	Fushui County	June 2008	20081001
13	Longan County	July 2008	20081002
14	Chongzhuo County	July 2008	20081003
15	Lanxu County	August 2008	20081004
16	Yongning County	September 2008	20081005
17	Shangsi County	October 2008	20081006
18	Lingshan County	November 2008	20081007
19	Mashan County	December 2008	20081008
20	Yongning Datang County	November 2009	20091001

^aAll samples were collected from the main place of production of Z. nitidum, Guangxi Province, China.

Figure 1. The structures of the alkaloids from the Z. nitidum roots. 1: nitidine chloride (NC); 2: ethoxychelerythrine; 3: liriodenine.

Table 2. Main factors and their level settings used for optimization of the extraction of the alkaloids.

Level	Extraction solvents (A)	Extraction number (B)	Extraction methods (C^a)	Volume of solvents (D, mL)
1	MeOH	2	Ultrasound	20
2	70% MeOH	3	Reflux	25
3	20%MeOH	4	SOAKING	30

^aAt room temperature, and the extraction times were 1 h.

Table 3. L₉ (3⁴) Matrix as the experimental design for optimization of the extraction of the three alkaloids.

	Column no.
Experimental trial	A^a	B^a	C^a	D^a	Total peak area of the alkaloids
1	1^b	1	1	1	9 223 528
2	1	2	2	2	10 112 133
3	1	3	3	3	7 827 213
4	2^b	1	2	3	10 504 364
5	2	2	3	1	5 663 974
6	2	3	1	2	8 977 292
7	3^b	1	3	2	1 244 095
8	3	2	1	3	1 348 094
9	3	3	2	1	1 597 171
	9 054 291.667	6 990 662.567	6 516 305.100	5 494 891.567	–
	8 381 876.867	5 708 067.200	7 404 556.233	6 777 840.100	–
	1 396 453.667	6 133 892.433	4 911 760.867	6 559 890.533	–
R^c	7 657 838.000	1 282 595.367	2 492 795.366	1 282 948.533	–

^aFour main factors.

^bThree levels for each main factor.

^cAverage response for each level.

Table 4. Analysis of variance.

Factors	Sum of squares of deviations	Degrees of freedom	F value	p Value^a	Significance
A	107 890 761 780 601.375	2	41.138	0.01 < p < 0.05	Yes
B (as error)	2 560 433 065 310.313	2	–	–	No
C	9 577 581 012 666.125	2	3.741	–	No
D	2 827 681 751 793.500	2	1.104	–	No

^aF_0.1(2,2) = 9.0; F_0.05(2,2) = 19.0; F_0.1(2,2) = 99.0.

Table 5. Regression equations and the respective correlation coefficients for quantitative determination of the three alkaloids by LC.

Reference substance	Correlation coefficient (r)	Regression equation^a	Linear range (μg/mL)
1	0.9995	y = 49386x−186 544	7.53–241.00
2	0.9999	y = 38902x−117 918	12.21–391.00
3	0.9995	y = 49563x−137 027	4.78–153.00

^ay denotes peak area, x denotes concentration of alkaloid compounds (μg/mL).

Table 6. Intra- and inter-day precision of the LC method for determination of the alkaloids.

	1		2		3
Precision	Peak area^a	RSD (%)	Peak area^a	RSD (%)	Peak area^a	RSD (%)
Intra-day^b	4 994 862 ± 38 135	0.76	5 756 849 ± 49362	0.82	883 397 ± 5322	0.60
Inter-day^c	5 006 658 ± 77 222	1.54	5 763 829 ± 35 549	0.62	882 514 ± 5988	0.68

^aMean ± SD.

^bSample analyzed five times on 1 day, n = 5.

^cSample analyzed each day on three consecutive days, n = 3.

Table 7. Stability and repeatability of the LC method for determination of the three alkaloids.

	1		2		3
Property	Peak area^a	RSD (%)	Peak area^a	RSD (%)	Peak area^a	RSD (%)
Stability	5 133 341 ± 68 626	1.34	5 755 657 ± 71 829	1.25	876 249 ± 10 461	1.19
Repeatability	5 117 142 ± 63 772	1.25	5 773 785 ± 47 219	0.82	87 7218 ± 11 929	1.36

^aMean ± SD, n = 3.

Table 8. Recoveries of the LC method for determination of the alkaloids.

Compounds	Concentration	Peak area^a	RSD (%)	Recovery (%)
1	Low	2 801 147 ± 126 99	0.45	101.59
	Medium	5 791 216 ± 13 312	0.99	101.63
	High	8 828 274 ± 108 314	1.23	102.17
2	Low	3 062 977 ± 40 327	1.32	98.42
	Medium	6 111 971 ± 59 926	0.98	96.38
	High	9 173 489 ± 104 608	1.14	95.83
3	Low	486 777 ± 52 241	1.07	100.21
	Medium	1 102 818 ± 7931	0.72	99.58
	High	1 657 730 ± 11 575	0.70	96.10

^aMean ± SD, n = 3.

Figure 2. LC profile obtained from a mixture of the three alkaloids: 1–3 = same as the structures in Figure 1.

Figure 3. Typical LC profile of an extract from the Z. nitidum roots (sample: 20): 1–3 = same as the structures in Figure 1.

Table 9. Alkaloids content of the Z. nitidum roots.

		Amount (mg/g crude drug)
No. Source		1	2	3	Total
1	Daxin County	1.120 ± 0.771	3.554 ± 1.409	0.819 ± 0.219	5.493
2	Qinzhou City	1.028 ± 0.327	2.838 ± 1.474	0.356 ± 0.154	4.222
3	Ningming County	1.059 ± 0.462	5.944 ± 1.161	0.374 ± 0.102	7.376
4	Tiandong County	1.294 ± 0.848	8.347 ± 3.288	0.331 ± 0.158	9.972
5	Wuming County	2.532 ± 1.337	4.685 ± 2.741	0.251 ± 0.085	7.468
6	Tiandeng County	2.689 ± 1.190	4.287 ± 1.804	0.348 ± 0.097	7.324
7	Jiuzhou Lingshan County	2.022 ± 0.345	5.746 ± 1.623	0.547 ± 0.145	8.315
8	Nanning City	1.644 ± 0.760	3.851 ± 2.340	0.325 ± 0.086	5.820
9	Baise City	1.058 ± 0.321	3.806 ± 1.322	0.309 ± 0.243	5.174
10	Bama County	2.271 ± 1.707	5.019 ± 3.459	0.266 ± 0.081	7.629
11	Napo County	3.377 ± 2.096	8.929 ± 5.780	0.544 ± 0.359	12.850
12	Fushui County	2.024 ± 1.029	6.143 ± 3.886	0.278 ± 0.140	8.445
13	Longan County	2.074 ± 0.940	4.326 ± 1.719	0.363 ± 0.186	6.763
14	Chongzhuo County	2.692 ± 1.442	4.584 ± 2.140	0.673 ± 0.299	7.949
15	Lanxu County	1.285 ± 0.575	4.448 ± 1.080	0.282 ± 0.089	6.015
16	Yongning County	1.812 ± 0.964	4.451 ± 0.564	0.349 ± 0.178	6.611
17	Shangsi County	2.931 ± 0.882	5.588 ± 1.017	0.374 ± 0.104	8.893
18	Shaping Lingshan County	2.868 ± 1.756	5.161 ± 2.449	0.401 ± 0.156	8.430
19	Mashan County	2.540 ± 1.433	3.269 ± 1.238	0.285 ± 0.053	6.094
20	Yongning Datang County	2.549 ± 1.001	6.162 ± 1.488	0.341 ± 0.200	9.051

1: Nitidine chloride (NC); 2: ethoxychelerythrine; 3: liriodenine.