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Table 1. Orthogonal test and results (L16, 4 4 ).
Test no. Ethanol (%)
Extraction
temperature(°C)
Liquid/ solid (ml/mg)
Extraction time
(min)
Mean yield
(%, n = 3)
1 50 30 1 30 2.65
2 50 50 1.5 40 3.05
3 50 60 2 50 2.51
4 50 70 2.5 60 2.54
5 60 30 1.5 50 3.08
6 60 50 1 60 3.25
7 60 60 2.5 30 3.15
8 60 70 2 40 2.82
9 70 30 2 60 2.46
10 70 50 2.5 50 3.38
11 70 60 1 40 2.91
12 70 70 1.5 30 2.65
13 80 30 2.5 40 2.28
14 80 50 2 30 2.24
15 80 60 1.5 60 2.12
16 80 70 1 50 1.93
K1 2.69 2.62 2.69 2.67
K2 3.08 2.98 2.73 2.77
K3 2.85 2.67 2.51 2.73
K4 2.14 2.49 2.84 2.59
R 0.94 0.49 0.33 0.18
P value 0.002 ** 0.011 * 0.037 * 0.170
* Significant (P < 0.05); ** very significant (P < 0.01).
analyzed. These factors are: sunshine (kh/y), precipitation (mm/y),
cumulative temperature (≥10°C/y), mean annual temperature
(°C/y), frost-free period (d/y), and altitude (m) were collected from
local weather records of producing areas, respectively. Soil factors
as soluble salinity (%), SOM (soil organic matter, %), Soil available
nitrogen, phosphorus and potassium were analyzed according to
the method of Täumer et al. (2005). The factors as sunshine,
precipitation, cumulative temperature, mean annual temperature,
frost-free period, altitude, soluble salinity and SOM were analyzed
by principle factors analysis with SPSS 16.0 to investigate
correlations between these environmental factors and the main
active components, because these are environmental factors
deciding the genuineness of L. barbarum fruits. While soil available
nitrogen, phosphorus and potassium can be easily changed by
human farming, so the available nitrogen, phosphorus and
potassium were analyzed by linear regression independently.
RESULTS
Preparation of the samples
A four-level OAD with an OA16 (4 4 ) matrix was chosen to
optimize the UAE parameters (Table 1). Based on singlefactor
experiments (Figure 1), the levels are chosen as:
ethanol concentration of 50/60/70/80%, extraction
temperature of 30/50/60/70°C, ratio of liquid to solid of
1/1.5/2/2.5, extraction time of 30/40/50/60 min. The
results of orthogonal test and extreme difference analysis
are presented in Table 1. The P value analysis indicated
that the influential order of the four factors on the
extraction yield of polyphenols is ethanol concentration >
extraction temperature > ratio of liquor to solid >
ultrasonication time (Table 1). According to variance
analysis, the contributions of ethanol concentration,
extraction temperature and ratio of liquor to solid for the
extraction yield of polyphenols are significant (P < 0.05),
whereas extraction time was not significant factor which
means UAE can significantly shorten extraction time.
According to extreme difference analysis, the optimum
extraction condition of polyphenols was deduced as:
ethanol of 70%, liquid/solid of 1/2.5 (ml/mg), extraction
temperature of 50°C, extraction time of 30 min.
Accuracy of determination on polyphenols from UAE
extracts
The linearity range of standard rutin was determined as
4.0 to 15.0 µg/ml (R 2 = 0.9998). The equation was
obtained by linear regression: y = 0.0419x - 0.1015 (data
not shown), where y is absorbance, x is concentration of
rutin (µg/ml). Recovery was obtained by adding standard
rutin to extract solution to obtain total polyphenols. Final
concentrations were 6.0, 11.0 and 15.0 µg/ml. The assay