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Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique
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Ultrasonics Sonochemistry 15 (2008) 376–385 www.elsevier.com/locate/ultsonch
Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique Hua-Feng Zhang a
a,b
, Tian-Shun Yang a, Zuo-Zhou Li a, Ying Wang
a,*
Key Laboratory of Plant Conservation Genetics, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, PR China b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Received 2 July 2007; received in revised form 16 August 2007; accepted 3 September 2007 Available online 12 September 2007
Abstract An ultrasonic-assisted extraction (UAE) procedure of epimedin A, epimedin B, epimedin C and icariin from Herba Epimedii was developed. The effects of ethanol concentration, ratio of liquid to solid, UAE time, extraction temperature and number of extraction cycles on the extraction yields of the four flavonoids from Herba Epimedii were investigated. The optimal UAE condition was found using orthogonal test: 50% (v/v) ethanol solution, liquid:solid ratio of 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C and three extraction cycles. The UAE method showed a high reproducibility. Epimedin A, B, C and icariin in the crude extract exhibited photodegradation under ultraviolet irradiation. This UAE method was shown to be highly efficient compared with the conventional Soxhlet extraction and boiling extraction. The effect of ultrasound on cell destruction was examined by scanning electron microscopy. The contents of epimedin A, B, C and icariin in the leaves of 20 Epimedium species were determined using high-performance liquid chromatographic method following UAE method. 2007 Elsevier B.V. All rights reserved. PACS: 43.35.Ei; 43.35.Vz; 87.50.Y Keywords: Extraction; Herba Epimedii; Ultrasound; Photostability
1. Introduction Herba Epimedii, the aerial parts of species of Epimedium L. (Berberidaceae), is one of the most famous Chinese herbal medicines listed in the Pharmacopoeia of the People’s Republic of China [1]. Herba Epimedii has been utilized to treat cardiovascular diseases and other chronic illness (infertility, amnesia and neurasthenia, impotence, senile functional diseases, etc.) in China for over 2000 years [2]. It is also grown as a herb for various medicinal purposes in Japan, Korea and the Mediterranean region [3,4]. The major active constituents of Herba Epimedii are flavonoids and more than 60 kinds of flavonoids have been identified
*
Corresponding author. Tel./fax: +86 27 87510675. E-mail address: [email protected] (Y. Wang).
1350-4177/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2007.09.002
[5–7]. Among them, epimedin A, B, C and icariin are considered major bioactive components that make up more than 52% of total flavonoids in Herba Epimedii [8]. Epimedin B, C and icariin were found being able to promote the proliferation of the osteoblast-like cells and having potential activity against osteoporosis [2]. Recent researches also demonstrated that epimedin A, C and icariin exhibit antitumor and immunoregulatory effects [6,9–12]. Epimedin A and icariin are observed to improve the function of the cardiovascular system [6,12]. Besides, icariin is considered to be a potential source of antioxidants, antidepressants and antiobesity agents [3,13,14]. Therefore, epimedin A, B, C and icariin possess important pharmacological activities and may become promising phytopharmaceuticals or nutraceuticals. It is of great interest to extract these medicinally active constituents from Herba Epimedii simultaneously.
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385
Extraction and determination of epimedin A, B, C and icariin in Herba Epimedii are also essential for the quality evaluation of the traditional Chinese medicine. A large number of studies found that the quality of Herba Epimedii often varied considerably according to its species, harvest season, storage, geographic origin or other growing conditions [15–17]. Epimedin A, B, C and icariin are usually used as the marker compounds for evaluating Herba Epimedii by high-performance liquid chromatography (HPLC) [8,11,17] and capillary zone electrophoresis [18]. Though determining the contents of epimedin A, B, C or icariin have been widely described [8,18], no study has been reported on the extraction of the four flavonoids simultaneously. Conventional Soxhlet extraction and boiling extraction are very time-consuming and require relatively high extraction temperature, which have been used for several decades. Thus, there is an increasing demand for a novel extraction technique with shortened extraction time, reduced extraction temperature, and increased extraction efficiency. Ultrasonic technique is being used widely in analytical chemistry, facilitating different steps in the analytical process, particularly in sample preparation [19–21]. Ultrasonic-assisted extraction (UAE) is an expeditious, inexpensive and efficient alternative to traditional extraction techniques and, in some cases, even to supercritical fluid and microwave-assisted extraction, which has been demonstrated by application to both organic and inorganic analytes in a wide variety of samples [22]. UAE of icariin from Herba Epimedii has been performed, but the recovery of icariin was not satisfactory [23]. Although total flavonoids of Herba Epimedii have also been extracted by means of ultrasonication, the recoveries of the four marker flavonoids (i.e. epimedin A, B, C and icariin) have not been determined [24]. Actually, total flavonoids are composed of not only bioactive constituents (e.g., icariin), but also various pigments without significant medicinal activities [15]. So far, there is no report for the simultaneous extraction of epimedin A, B, C and icariin using ultrasonic technique. The goals of this study are to optimize the UAE condition for extracting epimedin A, B, C and icariin from Herba Epimedii, compare UAE with boiling extraction and Soxhlet extraction, characterize the photostability of these four flavonoids in the crude extract, and measure the contents of these four flavonoids in 20 Epimedium species by HPLC method coupled with UAE method. 2. Experimental 2.1. Reagents and materials Standards of epimedin A, B, C and icariin were purchased from ChromaDex (USA). Ethanol and glacial acetic acid of analytical grade (Tianjin Bodi Chemicals Co. Ltd., China) were filtrated through 0.45 lm microporous membranes before use. Acetonitrile of HPLC grade (Fisher Chemicals, USA), ultrapure water obtained from a Milli-Q Plus system (Millipore, USA), and macroporous adsorp-
377
tion resin (Shandong Dongda Chemical Industry Company, China) were also used in this study. A total of 20 species of Epimedium L. were collected in the wild at the full blooming stage (Table 1). Vouched specimens were deposited at the Herbarium of Wuhan Botanical Garden, Chinese Academy of Sciences. Only Epimedium brevicornu collected from Shaanxi Province of China was used in the extraction experiments and the stability tests. The fresh leaves were harvested and dried in the shade with active ventilation at room temperature until constant weight. Dried leaves were then milled to an average size of 0.355 mm in diameter. The powdered samples were oven-dried at 65 C for 4 h, and then kept in a dry and dark place until use. 2.2. Equipments An ultrasonic cleaning bath (Wuhan Jiapeng Electronics Co. Ltd., China) with a frequency of 28 kHz and a maximum peak power of 500 W was used in this study. The ultrasound power actually delivered to each conical flask containing the ground sample–solvent mixture was measured to be 7 W using the calorimetric method. The HPLC system consisted of Agilent ChemStation Rev.A.10.02, G1313A autosampler, G1311A quarternary pump, G1314A variable wavelength ultraviolet (UV) detector (VWD), and reverse phase ZORBAX SB-C8 column (5 lm, 4.6 · 250 mm, Agilent Technologies, USA). Scanning electron micrographs were obtained using a Hitachi S-450 scanning electron microscope (Japan). A 200 W UV lamp (Shanghai Keyi Optical Instrument Company, China), emitting at 365 nm, was used for the photostability experiment. 2.3. Extraction method 2.3.1. UAE method Ground samples of 1.0 g were mixed with the appropriate extraction solvent in a 100 ml conical flask that was immersed in water in the ultrasonic cleaning bath. The bottom of the flask was approximately 2 cm above that of the bath and the liquid level in the flask was about 5 mm below the water surface in the bath. The flask containing the ground sample–solvent mixture was shaken every 5 min to ensure homogenous exposure of the mixture to ultrasound. The mixture was sonicated for specific time (10/ 20/30/40/50/60/70/80/90/100/120 min) at different temperature (25/35/40/45/50/55/65 C), and centrifuged at 3250g for 10 min. The supernatants were filtered through 0.45 lm microporous membranes and the filtrates were collected as the crude extracts. The crude extracts were then separated using macroporous adsorption resin and purified by the liquid–solid extraction system as described [25]. 2.3.2. Conventional extraction method Conventional extraction method included boiling extraction and Soxhlet extraction. Boiling extraction was
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Table 1 Determination of epimedin A, B, C and icariin in different Epimedium species using ultrasonic-assisted extraction method Taxon
Origin (locality)
Content (mg/g dried weight)a,b Epimedin A
Epimedin B
Epimedin C
Icariin
E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
Shaanxi, China South Korea Chongqing, China Zhejiang, China Guizhou, China Guizhou, China Hunan, China Guizhou, China Sichuan, China Guizhou, China Hunan, China Sichuan, China Hunan, China Chongqing, China Sichuan, China Sichuan, China Sichuan, China Yunnan, China Guizhou, China Hunan, China
5.44 2.03 4.39 7.03 1.95 0.89 n.f. 1.22 1.63 n.f. 4.14 1.90 1.56 n.f. 0.89 2.19 2.21 1.15 1.86 1.60
30.91 5.78 8.96 14.31 4.52 6.10 n.f. 2.70 4.32 0.68 9.54 5.19 3.90 n.f. 2.37 6.27 5.56 3.24 4.65 3.73
8.50 11.98 31.13 42.64 35.23 41.33 <0.50 37.90 12.05 106.99 12.06 18.91 11.44 <0.50 17.94 11.60 10.26 22.47 33.72 138.50
13.84 6.24 4.58 7.89 3.87 24.64 n.f. 2.94 2.44 <0.50 2.67 7.20 2.03 n.f. 3.06 8.64 3.00 3.14 4.68 2.73
a b
brevicornu Maxim. koeanum Nakai wushanense T.S. Ying sagittatum Maxim. acuminatum Franch. boreali-guizhouense S.Z. He and Y.K. Yang baojingense Q.L. Chen and B.M. Yang dewuense S.Z. He, Probst and W.F. Xu ecalcaratum G.Y. Zhong elachyphyllum W.T. Stearn epsteinii W.T. Stearn flavum W.T. Stearn hunanense (Hand.-Mazz.) Hand.-Mazz. ilicifolium W.T. Stearn membranaceum K. Meyer platypetalum K. Meyer reticulatum C.Y. Wu rhizomatosum W.T. Stearn shuichengense S.Z. He truncatum H.R. Liang
Values indicate the average of three replicates for each sample (RSDs < 3%). n.f., not found.
modified from Chen et al. [26]. Ground samples of 1.0 g were mixed with 50% (v/v) ethanol solution of 50 ml, steeped for 1 h, extracted for 30 min at 90 C, and filtrated through 0.45 lm microporous membrane. The filtrates were collected and the solids were extracted repeatedly (three times) in 50 ml volume of 50% ethanol solution. Soxhlet extraction was conducted according to a published method [27]. Briefly, ground samples of 1.0 g mixed with 100 ml of 50% ethanol solution were placed in a classical Soxhlet extraction apparatus at 90 ± 2 C. The extraction was performed for 90 and 240 min, respectively. 2.3.3. Extraction yield determination The extraction efficiency of UAE method and conventional extraction method was evaluated using the extraction yield as index, which was calculated according to the following equation: Y ¼ af =at where Y, af and at were the extraction yield of individual flavonoid (mg/g), the amount of individual flavonoid extracted (mg) and the total amount of Herba Epimedii used for the extraction (g), respectively. 2.3.4. Optimization of UAE parameters For optimization of UAE of epimedin A, B, C and icariin from Herba Epimedii, the effect of changing single factor on the extraction efficiencies of the four flavonoids was studied at first. The factors investigated in this report were as follows: ethanol concentration (0/10%/30%/50%/70%/ 90% v/v), ratio of liquid to solid (5/15/25/45/55 ml/g), ultrasonication duration (10/20/30/40/60/70/80/90/100/ 120 min) and extraction temperature (25/35/45/55/65 C).
Then, the extraction parameters of ultrasonication (i.e. ethanol concentration, ratio of liquid to solid, UAE time and extraction temperature) were optimized by an orthogonal test and further validated by a verification test. The investigated levels of each factor were selected depending on the results of single factor experiments. Multiple sequential extraction (3 · 30 min) of the same ground sample was performed to optimize and validate the number of extraction cycles. Ground sample was sonicated for 30 min and filtered through 0.45 lm microporous membrane. The filtrate was collected and the residue was subsequently extracted twice with the same volume of fresh solvent for the same extraction time. The actual content of individual flavonoid in ground sample was defined as the amount measured by Soxhlet extraction. The amount of individual flavonoid extracted in each ultrasonication cycle was expressed as a percentage of this content (i.e. recovery). In this study, each experiment was performed in triplicate and repeated at least once. 2.4. HPLC analysis Acetonitrile (solvent A) and 1.0% acetic acid (solvent B), deaerated ultrasonically for more than 30 min in advance, were used as the mobile phase with a flow rate of 1.0 ml/ min. The column temperature was 25 C and the sample volume injected was 5 ll. The detector was set at 270 nm. For the elution program, the following proportions of solvent A were used: 0–10 min, 20% solvent A; 10– 40 min, 20–25% solvent A; 40–42 min, 25–50% solvent A; 42 min, 50–20% solvent A. The chromatographic peaks of epimedin A, B, C and icariin were confirmed by comparing their retention time and UV spectra with those of the
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385 30
25
Extraction yield (mg/g)
15
10
25
Extraction yield (mg/g)
epimedin A epimedin B epimedin C icariin
20
5
0
20 epimedin A epimedin C
epimedin B Icariin
15 10 5 0
0
10
30
50
70
90
0
20
Ethanol concentration (%)
40
60
80
100
120
140
Extraction time (min)
Fig. 1. Effect of ethanol concentration on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ratio of liquid to solid: 100 ml/g; UAE time: 90 min; extraction temperature: 25 C; number of extraction cycles: 1.
Fig. 3. Effect of ultrasonic-assisted extraction time on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; ratio of liquid to solid: 30 ml/g; extraction temperature: 25 C; number of extraction cycles: 1.
35 epimedin A epimedin B epimedin C icariin
25 20 15 10
Extraction yield (mg/g)
30
Extraction yield (mg/g)
379
28
epimedin A by UAE
epimedin B by UAE
epimedin C by UAE epimedin A by BE
icariin by UAE epimedin B byBE
epimedin C by BE
icariin by BE
21
14
7 5
0 0 5
15
25
45
55
25
Ratio of liquor to solid (ml/g) Fig. 2. Effect of ratio of liquid to solid on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; UAE time: 90 min; extraction temperature: 25 C; number of extraction cycles: 1.
reference standards. Quantification was carried out by the integration of the peak using external standard method. 2.5. Scanning electron micrographs After extraction, the sample was centrifuged at 3250g for 10 min. The supernatant was discarded and the precipitate was dried. To determine the changes of particle size, the remaining residue was sputtered with gold and then examined by scanning electron microscopy under the high vacuum condition and at an operating voltage of 20 kV. To observe the cell damage, the residue was plunged in liquid nitrogen and then cut with a cold knife. The sectioned particles were sputtered with gold and examined by scanning electron microscopy [28].
35
45
55
65
Extraction temperature (°C) Fig. 4. Effect of extraction temperature on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; ratio of liquid to solid: 30 ml/g; UAE time: 30 min; number of extraction cycles: 1; UAE, ultrasonic-assisted extraction; BE, boiling extraction at different temperature.
2.6. Stability test 2.6.1. Stability of ultrasonication method Five replicates of the same ground sample (1.0 g) were processed according to the same optimal UAE protocol. The crude extracts were analyzed by HPLC and the extraction yields of epimedin A, B, C and icariin were calculated. The stability of ultrasonication method was assessed by comparing the results among the five replicates.
2.6.2. Photostability of epimedin A, B, C and icariin in the crude extract The photodegradation of epimedin A, B, C and icariin in the crude extract (aqueous ethanol solution) was carried
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out in quartz cells (optical path 10 mm) where the crude extract was irradiated with a UV lamp for 0–22 h at 25 ± 2 C. In order to avoid overheating of the samples, a cooling fan and an air-conditioner were employed to eliminate the heat from the UV lamp and to control the room temperature, respectively. The distance between the cells and the lamp was 5 cm. Samples were taken at 0, 2, 3, 12, 16, 18, 22 h and the concentrations (ng/ll) of the four flavonoids of each sample were monitored by HPLC. The photodegradation rate of individual flavonoid was calculated according to the following formula: R ¼ ðc0 c1 Þ=c0 100% where R, c0 and c1 were the photodegradation rate (%), the concentration (ng/ll) of individual flavonoid in the initial crude extract prior to UV irradiation and the concentration (ng/ll) of individual flavonoid in the degraded crude extract at different UV radiation time, respectively. 3. Results and discussion 3.1. Optimization of parameters for UAE 3.1.1. Effects of extraction variables Both methanol and ethanol have been successfully used as the solvent for extracting icariin and total flavonoids in Herba Epimedii [23]. However, methanol was regarded as not in compliance with good manufacturing practice (GMP) due to its high toxicity [27]. Ethanol was thus adopted as the solvent for UAE in this study. Fig. 1 shows that the extraction yields of epimedin A, B, C and icariin with aqueous ethanol solution as the solvent are higher than those with water (0% ethanol solution) as the solvent. The results also indicated that the extraction yields of the four flavonoids first increased and then decreased with
the increase of ethanol concentration, with the highest yields in 50% (v/v) ethanol solution. The effects of liquid:solid ratio on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii are shown in Fig. 2. The results demonstrated that the extraction yield of epimedin C rose as liquid:solid ratio increased. When the ratio increased from 15 to 25 ml/g, the yields of epimedin A, B and icariin increased dramatically. In order to obtain more target flavonoids with less solvent, the ratio of liquid to solid should be maintained in the range of 20– 45 ml/g. The influence of UAE time on the extraction of epimedin A, B, C and icariin is shown in Fig. 3. The results indicated that the extraction yields of the four flavonoids increased with the increase of ultrasonication duration. There is an obvious increasing trend for the four flavonoids. For epimedin B, C and icariin, the yield increased rapidly at 10–30 min and then slowly after 30 min. The extraction yield of epimedin A was much lower than the others. But the yield of epimedin A still increased slightly at 10–30 min, and reached the plateau after 40 min. The impacts of extraction temperature on the extraction yields of epimedin A, B, C and icariin are demonstrated in Fig. 4. When the temperature was increased from 25 C to 65 C, the extraction yield of icariin was improved. However, the yield of epimedin B was not significantly affected by extraction temperature. The yields of epimedin A and C rose within the first 45 C, after which the yields declined. The high yields of target flavonoids at elevated temperature may be partially attributed to the increase in the number of cavitation bubbles formed by ultrasound and to the enhanced mass transfer rates [20]. Nevertheless, when temperature rose to a certain degree (i.e. above 45 C), the yields of epimedin A and C decreased. One of the possible reasons for this phenomenon is that epimedin A and C suffered thermodegradation, but the contents of epimedin A,
Table 2 Orthogonal test design and results Test no.
Factor
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a1 a1 a1 a1 a2 a2 a2 a2 a3 a3 a3 a3 a4 a4 a4 a4
(a) Ethanol concentration (%) 30 30 30 30 40 40 40 40 50 50 50 50 60 60 60 60
Extraction yield (mg/g) (b) Ratio of liquid to solid (ml/g) b1 b2 b3 b4 b1 b2 b3 b4 b1 b2 b3 b4 b1 b2 b3 b4
20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35
(c) Ultrasonic-assisted extraction time (min) c1 c2 c3 c4 c2 c1 c4 c3 c3 c4 c1 c2 c4 c3 c2 c1
20 30 40 50 30 20 50 40 40 50 20 30 50 40 30 20
(d) Extraction temperature (C) d1 d2 d3 d4 d3 d4 d1 d2 d4 d3 d2 d1 d2 d1 d4 d3
40 45 50 55 50 55 40 45 55 50 45 40 45 40 55 50
Epimedin A
Epimedin B
Epimedin C
Icariin
2.2815 3.5943 4.6311 2.7273 4.1845 3.3888 3.8447 3.5100 2.2933 3.0040 4.0025 3.7126 2.5439 2.4010 4.7862 3.2102
22.3098 23.4166 21.3263 22.0101 22.0432 19.7327 19.0011 20.7453 19.5211 20.6122 19.8870 23.9842 18.8712 18.4276 20.9011 21.8392
5.6163 5.8701 7.3727 5.6204 6.5525 5.7325 6.0219 5.7021 6.1033 6.5430 7.4888 6.1138 5.7000 5.6003 6.3020 5.8796
6.1098 8.9707 11.1315 10.5222 11.3012 8.3264 7.0303 10.6213 10.1999 12.0002 10.2096 10.9877 7.8314 6.4726 9.3021 12.6000
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385
B, C and icariin in the crude extract did not declined with the increase of temperature (up to 65 C) without ultrasound (data not shown). To gain further insight into the phenomenon, boiling extraction was modified and performed at different temperature (25/35/45/55/65 C). As shown in Fig. 4, the yields of the four flavonoids obtained by boiling extraction increase with the increase of temperature, which does not indicate thermal instability of the four flavonoids. It is also possible that extremely high temperature decreased the number of cavitation bubbles [29] and weakened the impact of cavity collapse on ground samples, which resulted in the decline of the yields of epimedin A and C. Furthermore, extremely high temperature might lead to some other disadvantages, such as increase of energy cost, acceleration of solvent volatilization and enhancement of impurities extraction. Hence, extraction temperature should not exceed 55 C. Table 3 Extreme difference analysis of orthogonal test Statistical item
Flavonoid
Factor a
b
c
d
381
3.1.2. Results and analysis of orthogonal test In order to obtain an optimal condition under which the extraction yields of epimedin A, B, C and icariin were all high, an orthogonal test was designed to optimize parameters on the basis of the results of single factor experiments (see above). Four factors, ethanol concentration (a), ratio of liquid to solid (b), UAE time (c) and extraction temperature (d), were selected for optimization, each at four levels (Table 2). The extraction yields of the four flavonoids were regarded as investigated indexes. The total evaluation indexes were analyzed by statistical method, i.e. extreme difference analysis and variance analysis. The results of orthogonal test, extreme difference analysis and variance analysis are presented in Tables 2–4, respectively. The analysis of extreme difference revealed that the influential order of the four factors on the extraction yields of epimedin A, B, C and icariin is b > c > d > a, c > a > b > d, b > d > a > c and d > b > a > c, respectively. This order was in agreement with that based on the F values in variance analysis. In addition, according to variance analysis, the contributions of b and c were significant for the extraction yield of epimedin A (P < 0.05). For the extraction yield of epimedin B, all factors except d showed significant influence (P < 0.05), which confirmed the results of single factor experiments. The effects of a, b and d on the yield of icariin were very significant (P < 0.01) and effect of
T1
Epimedin A Epimedin B Epimedin C Icariin
13.2342 89.0628 24.4795 36.7342
11.3032 82.7453 23.9721 35.4423
12.8830 83.7687 24.7172 37.2458
12.2398 83.7227 23.3523 30.6004
T2
Epimedin A Epimedin B Epimedin C Icariin
14.9280 81.5223 24.0090 37.2792
12.3881 82.1891 23.7459 35.7699
16.2776 90.3451 24.8384 40.5617
13.6507 82.9201 24.7610 37.6330
Table 4 Variance analysis of orthogonal test
Epimedin A Epimedin B Epimedin C Icariin
13.0124 84.0045 26.2489 43.3974
17.2645 81.1155 27.1854 37.6735
12.8354 80.0203 24.7784 38.4253
15.0298 85.8209 26.3478 47.0329
a
Epimedin A Epimedin B Epimedin C Icariin
12.9413 80.0391 23.4819 36.2061
13.1601 88.5788 23.3159 44.7312
12.1199 80.4946 23.8853 37.3841
13.1956 82.1650 23.7582 38.3506
Epimedin A Epimedin B Epimedin C Icariin
3.3086 22.2657 6.1199 9.1836
2.8258 20.6863 5.9930 8.8606
3.2208 20.9422 6.1793 9.3115
3.0600 20.9307 5.8381 7.6501
Epimedin A Epimedin B Epimedin C Icariin
3.7320 20.3806 6.0023 9.3198
3.0970 20.5473 5.9365 8.9425
4.0694 22.5863 6.2096 10.1404
3.4127 20.7300 6.1903 9.4083
Epimedin A Epimedin B Epimedin C Icariin
3.2531 21.0011 6.5622 10.8494
4.3161 20.2789 6.7964 9.4184
3.2089 20.0051 6.1946 9.6063
3.7575 21.4552 6.5870 11.7582
Epimedin A Epimedin B Epimedin C Icariin
3.2353 20.0098 5.8705 9.0515
3.2900 22.1447 5.8290 11.1828
3.0300 20.1237 5.9713 9.3460
3.2989 20.5413 5.9396 9.5877
Epimedin A Epimedin B Epimedin C Icariin
0.4967 2.2559 0.6917 1.7979
1.4903 1.8658 0.9674 2.3222
1.0394 2.5812 0.2383 0.8289
0.6975 0.9139 0.7489 4.1081
T3
T4
K1
K2
K3
K4
R
Sourcea Flavonoid
Mean square
Epimedin A 0.664 Epimedin B 11.747 Epimedin C 1.081 Icariin 8.455
3 3 3 3
0.221 3.916 0.360 2.818
2.793 18.260 3.465 105.246
Epimedin A 5.086 Epimedin B 8.417 Epimedin C 2.362 Icariin 14.069
3 3 3 3
1.695 2.806 0.787 4.690
21.393 13.084 7.570 175.141
*
Epimedin A 2.610 Epimedin B 16.992 Epimedin C 0.151 Icariin 1.759
3 3 3 3
0.870 5.664 0.050 0.586
10.976 26.413 0.485 21.902
*
Epimedin A 1.010 Epimedin B 1.864 Epimedin C 1.334 Icariin 33.988
3 3 3 3
0.337 0.621 0.445 11.329
4.248 2.897 4.276 423.096
Error
Epimedin A Epimedin B Epimedin C Icariin
0.238 0.643 0.312 0.080
3 3 3 3
0.079 0.214 0.104 0.027
Total
Epimedin A 9.608 Epimedin B 39.663 Epimedin C 5.240 Icariin 58.351
15 15 15 15
c
d
F
Significanceb
d.f.
b
Sum of squares
*
**
*
**
*
*
**
a a, Ethanol concentration (%); b, ratio of liquid to solid (ml/g); c, UAE time (min); d, extraction temperature (C). b * , Significant (P < 0.05); **, very significant (P < 0.01).
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epimedin A, B, C and icariin in Herba Epimedii by HPLC or capillary zone electrophoresis.
Table 5 Results of verification test Test no.
Factor
Extraction yield (mg/g)
a
b
c
d
Epimedin A
Epimedin B
Epimedin C
Icariin
3.2. Reproducibility of ultrasonication method
17 18 19 20
a2 a1 a3 a3
b3 b4 b3 b4
c2 c2 c2 c2
d3 d3 d3 d3
5.0691 4.2168 4.9032 4.1967
22.8790 28.2213 25.7069 25.6989
6.9089 6.4330 7.5743 6.7223
11.5310 11.8363 12.8466 12.8300
To study the stability of UAE method for epimedin A, B, C and icariin in Herba Epimedii, five ground samples of 1.0 g were processed under the optimized condition, i.e. ethanol concentration of 50%, liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C in three extraction cycles (data not shown). The relative standard deviations (RSDs) for the determined extraction yields of the four flavonoids are found to be less than 3% (n = 5), showing a good reproducibility of UAE method.
c was significant (P < 0.05). However, none of the four factors significantly affected the yield of epimedin C. According to extreme difference analysis, the optimum conditions for extracting epimedin A, B, C and icariin were a2b3c2d3, a1b4c2d3, a3b3c2d3 and a3b4c2d3, respectively. However, all of these conditions were not included in orthogonal test. In order to further validate these optimal conditions, UAE under these conditions was implemented. The results of verification test are listed in Table 5. The highest yields of epimedin C and icariin were obtained under the condition of a3b3c2d3 (Tables 2 and 5). In fact, the yields of epimedin A and B were also high under this condition. So this condition was rationally confirmed to be the most optimum, namely, ethanol concentration of 50% (v/v), liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C.
3.1.3. Validation of number of extraction cycles In general, the extraction amount of constituents increases with the increase of the extraction cycles. In order to investigate the influence of number of extraction cycles on the recoveries of epimedin A, B, C and icariin, ground sample was extracted triplicately (3 · 30 min) under the above optimized condition (a3b3c2d3). The results are listed in Table 6. The recoveries of the four flavonoids slightly increased with the increase of the extraction cycles. After the third extraction cycle, 100% of the four flavonoids were recovered. Therefore, three ultrasonication cycles were performed in sample preparation for the determination of
3.3. Photostability of flavonoids In plants, flavonoids are reported resistant to heat, oxygen, dryness and moderate degrees of acidity but prone to be modified by short wavelength light [30]. The concentrations of epimedin A, B, C and icariin in the crude extract (ethanol solution) decreased as UV irradiation time was extended (data not shown). When epimedin A, B, C and icariin were subjected to UV radiation for 22 h, their photodegradation rates reached 15.16%, 4.69%, 4.73% and 3.78%, respectively. However, no significant photodegradation of the four flavonoids in the crude extract stored in the shade for about 7 days was observed (data not shown). Hence, short wavelength light should be avoided for the extraction and storage of Herba Epimedii samples. 3.4. Comparison of UAE and conventional extraction method The extraction efficiency of UAE was compared with that of other classical methods, Soxhlet extraction and boiling extraction. Table 6 summarizes the results. After extraction for 90 min, the best recoveries (100%) of epimedin A, B, C and icariin were achieved by UAE at the
Table 6 Recoveries of epimedin A, B, C and icariin versus number of extraction cycles in different extraction methods Extraction method
Number of extraction cycles
Ultrasonicassisted extraction
1 2 3
30 60 90
50 C
Boiling extraction
1 2 3 4
90 120 150 180
Soxhlet extraction
1 1
90 240
a b
Extraction time (min)
Extraction temperature
Amount of solvent (ml)a
Recovery (%)b Epimedin A
Epimedin B
Epimedin C
Icariin
30 60 90
93.4 100.0 –
92.6 100.0 –
91.5 99.8 100.0
90.9 99.4 100.0
90 C
50 100 150 200
83.1 95.4 98.7 100.0
96.7 99.1 100.0 –
35.8 82.0 96.0 100.0
69.8 94.4 99.1 100.0
90 ± 2 C
100 100
41.6 100.0
44.8 100.0
37.0 100.0
40.0 100.0
Ground samples of 1.0 g are used in each extraction method. The recovery means the extracted target flavonoid in % related to its content in the studied Herba Epimedii, measured by classical Soxhlet method.
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Fig. 5. Scanning electron micrographs of Herba Epimedii leaf samples from different extraction methods at low magnification (100·, A–C) and high magnification (1500·, D–F). A and D, untreated leaf; B and E, after boiling extraction; C and F, after ultrasonication.
extraction temperature of 50 C, which were higher than those by boiling extraction at 90 C and Soxhlet extraction at 90 ± 2 C. After four extraction cycles at 90 C, boiling extraction used a total of 200 ml of 50% ethanol solution as the solvent for 180 min and obtained the same recoveries (100%) of the four flavonoids as UAE with 90 ml of ethanol solution for 90 min at 50 C. After 240 min extraction
at 90 ± 2 C, Soxhlet extraction using 100 ml of 50% ethanol solution as the solvent gave the same efficiency (100%) as UAE with 90 ml of 50% ethanol solution for 90 min at 50 C. Clearly, by reducing the extraction time, the extraction temperature, and the quantity of solvent required, UAE was proven to be a robust method which can offer improved extractability.
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In order to understand the mechanism of UAE and to elucidate each extraction procedure, the leaf samples from different extraction methods (UAE and boiling extraction) were examined by scanning electron microscopy (Fig. 5). Low magnification (100·) micrographs (Fig. 5A–C) indicated that UAE reduced the particle size of Herba Epimedii leaf samples, which increased the number of cells directly exposed to the solvent and thus exposed to ultrasonically induced cavitation. However, boiling extraction did not clearly decrease the particle size. High magnification images at 1500· magnification (Fig. 5D–F) revealed that the surface of the leaf sample was seriously destroyed after UAE, while only some slight ruptures took place on the surface of the sample after boiling extraction. The solvent extraction from the dried leaf materials includes two stages: steeping leaf in solvent and mass transfer of soluble constituents from leaf to solvent [20]. Ultrasound can produce acoustic cavitation and facilitate the swelling and solvatation of the leaves causing enlargement of the pores on the cell surface, which will improve both the solvent penetration into the leaf materials and the release of the cell contents. This mechanism of UAE is different from those of boiling extraction and Soxhlet extraction that depend on a series of permeation and solubilization processes to wash the intracellular analytes out of the matrix [28]. 3.5. Application of UAE method to Epimedium samples from different origins Epimedin A, B, C and icariin of 20 Epimedium species from different origins were extracted under the optimal UAE condition and quantified by HPLC (Table 1). A large variation of the contents of the four flavonoids was found among these Epimedium species from different origins. The contents of epimedin A in the leaves of E. sagittatum, epimedin B in E. brevicornu, epimedin C in E. truncatum and icariin in E. boreali-guizhouense were the highest, respectively. The variation is possibly attributed to several factors, such as Epimedium species, soil and climate of the collection sites. 4. Conclusions The UAE method reported here can offer an effective alternative for simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii. It can be applied to sample preparation for the quality control of Herba Epimedii. Additionally, it might serve as a promising industrial extraction protocol of the four flavonoids. Compared with conventional boiling extraction and Soxhlet extraction, UAE is an easy, efficient and inexpensive method with low toxicity and high reproducibility. The optimal extraction condition is ethanol concentration of 50% (v/v), liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C and three extraction cycles. In the crude extract, epimedin A, B, C and icariin can be partially degraded with exposure to UV light.
The UAE method has been applied successfully to quantify epimedin A, B, C and icariin in the leaves of 20 Epimedium species coupled with HPLC method. The contents of the four flavonoids drastically varied among the leaves of these Epimedium species from different origins. Acknowledgements This research was partially funded by the National Natural Science Foundation of China (Grant No. 30570171), 100 Talents Program of Chinese Academy of Sciences (05045112) and the Directional Project of Chinese Academy of Sciences. The authors would like to thank Prof. Wang Yuchun from Institute of Process Engineering, Chinese Academy of Sciences for critical review of the manuscript. References [1] The State Pharmacopoeia Commission of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, vol. I, Chemical Industry Press, Beijing, 2005, p. 229. [2] F.H. Meng, Y.B. Li, Z.L. Xiong, Z.M. Jiang, F.M. Li, Phytomedicine 12 (2005) 189. [3] Y.K. Wang, Z.Q. Huang, Pharmacological Research 52 (2005) 174. [4] A.D. Naeyer, V. Pocock, S. Milligan, D.D. Keukeleire, Fitoterapia 76 (2005) 35. [5] R. Liu, A. Li, A. Sun, J. Cui, L. Kong, Journal of Chromatography A 1064 (2005) 53. [6] X. Zhang, Y. Li, X. Yang, K. Wang, J. Ni, X. Qu, Life Sciences 78 (2005) 180. [7] H. Wu, E.J. Lien, L.L. Lien, Progress in Drug Research 60 (2003) 1. [8] B. Guo, P. Xiao, Natural Product Research and Development 8 (1996) 74. [9] T.Z. Liu, C.Y. Chen, S.J. Yiin, C.H. Chen, J.T. Cheng, M.K. Shih, Y.S. Wang, C.L. Chern, Food and Chemical Toxicology 44 (2006) 227. [10] H.R. Liang, P. Vuorela, H. Vuorela, R. Hiltunen, Planta Medica 63 (1997) 316. [11] Y.B. Li, Z.L. Xiong, F.M. Li, Journal of Chromatography B 821 (2005) 235. [12] D. Zhu, L. Qu, X. Zhang, Y. Lou, European Journal of Pharmacology 514 (2005) 99. [13] Y. Pan, L. Kong, X. Xia, W. Zhang, Z. Xia, F. Jiang, Pharmacology, Biochemistry and Behavior 82 (2005) 686. [14] X. Zheng, Y. Hu, J. Liu, K. Ouyang, Bioorganic and Medicinal Chemistry Letters 15 (2005) 4531. [15] B. Guo, P. Xiao, Acta Phytotaxonomica Sinica 37 (1999) 228. [16] C.P. Chen, M. Sha, S.S. Tang, China Journal of Chinese Materia Medica 21 (1996) 86. [17] P. Shen, B.L. Guo, Y. Gong, D.Y.Q. Hong, Y. Hong, E.L. Yong, Phytochemistry 68 (2007) 1448. [18] J.J. Liu, S.P. Li, Y.T. Wang, Journal of Chromatography A 1103 (2006) 344. [19] S. Cabredo-Pinillos, T. Cedro´n-Ferna´ndez, M. Gonza´lez-Briongos, L. Puente-Pascual, C. Sa´enz-Barrio, Talanta 69 (2006) 1123. [20] M. Vinatoru, Ultrasonics Sonochemistry 8 (2001) 303. [21] L. Wang, C.L. Weller, Trends in Food Science and Technology 17 (2006) 300. [22] N. Jalbani, T.G. Kazi, B.M. Arain, M.K. Jamali, H.I. Afridi, R.A. Sarfraz, Talanta 70 (2006) 307. [23] F. Yang, G.X. Zhong, H.P. Zhao, C.Y. Lin, X.O. Wen, X.L. He, China Pharmacy 16 (2005) 416.
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Ultrasonics Sonochemistry 15 (2008) 376–385 www.elsevier.com/locate/ultsonch
Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique Hua-Feng Zhang a
a,b
, Tian-Shun Yang a, Zuo-Zhou Li a, Ying Wang
a,*
Key Laboratory of Plant Conservation Genetics, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, PR China b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Received 2 July 2007; received in revised form 16 August 2007; accepted 3 September 2007 Available online 12 September 2007
Abstract An ultrasonic-assisted extraction (UAE) procedure of epimedin A, epimedin B, epimedin C and icariin from Herba Epimedii was developed. The effects of ethanol concentration, ratio of liquid to solid, UAE time, extraction temperature and number of extraction cycles on the extraction yields of the four flavonoids from Herba Epimedii were investigated. The optimal UAE condition was found using orthogonal test: 50% (v/v) ethanol solution, liquid:solid ratio of 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C and three extraction cycles. The UAE method showed a high reproducibility. Epimedin A, B, C and icariin in the crude extract exhibited photodegradation under ultraviolet irradiation. This UAE method was shown to be highly efficient compared with the conventional Soxhlet extraction and boiling extraction. The effect of ultrasound on cell destruction was examined by scanning electron microscopy. The contents of epimedin A, B, C and icariin in the leaves of 20 Epimedium species were determined using high-performance liquid chromatographic method following UAE method. 2007 Elsevier B.V. All rights reserved. PACS: 43.35.Ei; 43.35.Vz; 87.50.Y Keywords: Extraction; Herba Epimedii; Ultrasound; Photostability
1. Introduction Herba Epimedii, the aerial parts of species of Epimedium L. (Berberidaceae), is one of the most famous Chinese herbal medicines listed in the Pharmacopoeia of the People’s Republic of China [1]. Herba Epimedii has been utilized to treat cardiovascular diseases and other chronic illness (infertility, amnesia and neurasthenia, impotence, senile functional diseases, etc.) in China for over 2000 years [2]. It is also grown as a herb for various medicinal purposes in Japan, Korea and the Mediterranean region [3,4]. The major active constituents of Herba Epimedii are flavonoids and more than 60 kinds of flavonoids have been identified
*
Corresponding author. Tel./fax: +86 27 87510675. E-mail address: [email protected] (Y. Wang).
1350-4177/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2007.09.002
[5–7]. Among them, epimedin A, B, C and icariin are considered major bioactive components that make up more than 52% of total flavonoids in Herba Epimedii [8]. Epimedin B, C and icariin were found being able to promote the proliferation of the osteoblast-like cells and having potential activity against osteoporosis [2]. Recent researches also demonstrated that epimedin A, C and icariin exhibit antitumor and immunoregulatory effects [6,9–12]. Epimedin A and icariin are observed to improve the function of the cardiovascular system [6,12]. Besides, icariin is considered to be a potential source of antioxidants, antidepressants and antiobesity agents [3,13,14]. Therefore, epimedin A, B, C and icariin possess important pharmacological activities and may become promising phytopharmaceuticals or nutraceuticals. It is of great interest to extract these medicinally active constituents from Herba Epimedii simultaneously.
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385
Extraction and determination of epimedin A, B, C and icariin in Herba Epimedii are also essential for the quality evaluation of the traditional Chinese medicine. A large number of studies found that the quality of Herba Epimedii often varied considerably according to its species, harvest season, storage, geographic origin or other growing conditions [15–17]. Epimedin A, B, C and icariin are usually used as the marker compounds for evaluating Herba Epimedii by high-performance liquid chromatography (HPLC) [8,11,17] and capillary zone electrophoresis [18]. Though determining the contents of epimedin A, B, C or icariin have been widely described [8,18], no study has been reported on the extraction of the four flavonoids simultaneously. Conventional Soxhlet extraction and boiling extraction are very time-consuming and require relatively high extraction temperature, which have been used for several decades. Thus, there is an increasing demand for a novel extraction technique with shortened extraction time, reduced extraction temperature, and increased extraction efficiency. Ultrasonic technique is being used widely in analytical chemistry, facilitating different steps in the analytical process, particularly in sample preparation [19–21]. Ultrasonic-assisted extraction (UAE) is an expeditious, inexpensive and efficient alternative to traditional extraction techniques and, in some cases, even to supercritical fluid and microwave-assisted extraction, which has been demonstrated by application to both organic and inorganic analytes in a wide variety of samples [22]. UAE of icariin from Herba Epimedii has been performed, but the recovery of icariin was not satisfactory [23]. Although total flavonoids of Herba Epimedii have also been extracted by means of ultrasonication, the recoveries of the four marker flavonoids (i.e. epimedin A, B, C and icariin) have not been determined [24]. Actually, total flavonoids are composed of not only bioactive constituents (e.g., icariin), but also various pigments without significant medicinal activities [15]. So far, there is no report for the simultaneous extraction of epimedin A, B, C and icariin using ultrasonic technique. The goals of this study are to optimize the UAE condition for extracting epimedin A, B, C and icariin from Herba Epimedii, compare UAE with boiling extraction and Soxhlet extraction, characterize the photostability of these four flavonoids in the crude extract, and measure the contents of these four flavonoids in 20 Epimedium species by HPLC method coupled with UAE method. 2. Experimental 2.1. Reagents and materials Standards of epimedin A, B, C and icariin were purchased from ChromaDex (USA). Ethanol and glacial acetic acid of analytical grade (Tianjin Bodi Chemicals Co. Ltd., China) were filtrated through 0.45 lm microporous membranes before use. Acetonitrile of HPLC grade (Fisher Chemicals, USA), ultrapure water obtained from a Milli-Q Plus system (Millipore, USA), and macroporous adsorp-
377
tion resin (Shandong Dongda Chemical Industry Company, China) were also used in this study. A total of 20 species of Epimedium L. were collected in the wild at the full blooming stage (Table 1). Vouched specimens were deposited at the Herbarium of Wuhan Botanical Garden, Chinese Academy of Sciences. Only Epimedium brevicornu collected from Shaanxi Province of China was used in the extraction experiments and the stability tests. The fresh leaves were harvested and dried in the shade with active ventilation at room temperature until constant weight. Dried leaves were then milled to an average size of 0.355 mm in diameter. The powdered samples were oven-dried at 65 C for 4 h, and then kept in a dry and dark place until use. 2.2. Equipments An ultrasonic cleaning bath (Wuhan Jiapeng Electronics Co. Ltd., China) with a frequency of 28 kHz and a maximum peak power of 500 W was used in this study. The ultrasound power actually delivered to each conical flask containing the ground sample–solvent mixture was measured to be 7 W using the calorimetric method. The HPLC system consisted of Agilent ChemStation Rev.A.10.02, G1313A autosampler, G1311A quarternary pump, G1314A variable wavelength ultraviolet (UV) detector (VWD), and reverse phase ZORBAX SB-C8 column (5 lm, 4.6 · 250 mm, Agilent Technologies, USA). Scanning electron micrographs were obtained using a Hitachi S-450 scanning electron microscope (Japan). A 200 W UV lamp (Shanghai Keyi Optical Instrument Company, China), emitting at 365 nm, was used for the photostability experiment. 2.3. Extraction method 2.3.1. UAE method Ground samples of 1.0 g were mixed with the appropriate extraction solvent in a 100 ml conical flask that was immersed in water in the ultrasonic cleaning bath. The bottom of the flask was approximately 2 cm above that of the bath and the liquid level in the flask was about 5 mm below the water surface in the bath. The flask containing the ground sample–solvent mixture was shaken every 5 min to ensure homogenous exposure of the mixture to ultrasound. The mixture was sonicated for specific time (10/ 20/30/40/50/60/70/80/90/100/120 min) at different temperature (25/35/40/45/50/55/65 C), and centrifuged at 3250g for 10 min. The supernatants were filtered through 0.45 lm microporous membranes and the filtrates were collected as the crude extracts. The crude extracts were then separated using macroporous adsorption resin and purified by the liquid–solid extraction system as described [25]. 2.3.2. Conventional extraction method Conventional extraction method included boiling extraction and Soxhlet extraction. Boiling extraction was
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Table 1 Determination of epimedin A, B, C and icariin in different Epimedium species using ultrasonic-assisted extraction method Taxon
Origin (locality)
Content (mg/g dried weight)a,b Epimedin A
Epimedin B
Epimedin C
Icariin
E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
Shaanxi, China South Korea Chongqing, China Zhejiang, China Guizhou, China Guizhou, China Hunan, China Guizhou, China Sichuan, China Guizhou, China Hunan, China Sichuan, China Hunan, China Chongqing, China Sichuan, China Sichuan, China Sichuan, China Yunnan, China Guizhou, China Hunan, China
5.44 2.03 4.39 7.03 1.95 0.89 n.f. 1.22 1.63 n.f. 4.14 1.90 1.56 n.f. 0.89 2.19 2.21 1.15 1.86 1.60
30.91 5.78 8.96 14.31 4.52 6.10 n.f. 2.70 4.32 0.68 9.54 5.19 3.90 n.f. 2.37 6.27 5.56 3.24 4.65 3.73
8.50 11.98 31.13 42.64 35.23 41.33 <0.50 37.90 12.05 106.99 12.06 18.91 11.44 <0.50 17.94 11.60 10.26 22.47 33.72 138.50
13.84 6.24 4.58 7.89 3.87 24.64 n.f. 2.94 2.44 <0.50 2.67 7.20 2.03 n.f. 3.06 8.64 3.00 3.14 4.68 2.73
a b
brevicornu Maxim. koeanum Nakai wushanense T.S. Ying sagittatum Maxim. acuminatum Franch. boreali-guizhouense S.Z. He and Y.K. Yang baojingense Q.L. Chen and B.M. Yang dewuense S.Z. He, Probst and W.F. Xu ecalcaratum G.Y. Zhong elachyphyllum W.T. Stearn epsteinii W.T. Stearn flavum W.T. Stearn hunanense (Hand.-Mazz.) Hand.-Mazz. ilicifolium W.T. Stearn membranaceum K. Meyer platypetalum K. Meyer reticulatum C.Y. Wu rhizomatosum W.T. Stearn shuichengense S.Z. He truncatum H.R. Liang
Values indicate the average of three replicates for each sample (RSDs < 3%). n.f., not found.
modified from Chen et al. [26]. Ground samples of 1.0 g were mixed with 50% (v/v) ethanol solution of 50 ml, steeped for 1 h, extracted for 30 min at 90 C, and filtrated through 0.45 lm microporous membrane. The filtrates were collected and the solids were extracted repeatedly (three times) in 50 ml volume of 50% ethanol solution. Soxhlet extraction was conducted according to a published method [27]. Briefly, ground samples of 1.0 g mixed with 100 ml of 50% ethanol solution were placed in a classical Soxhlet extraction apparatus at 90 ± 2 C. The extraction was performed for 90 and 240 min, respectively. 2.3.3. Extraction yield determination The extraction efficiency of UAE method and conventional extraction method was evaluated using the extraction yield as index, which was calculated according to the following equation: Y ¼ af =at where Y, af and at were the extraction yield of individual flavonoid (mg/g), the amount of individual flavonoid extracted (mg) and the total amount of Herba Epimedii used for the extraction (g), respectively. 2.3.4. Optimization of UAE parameters For optimization of UAE of epimedin A, B, C and icariin from Herba Epimedii, the effect of changing single factor on the extraction efficiencies of the four flavonoids was studied at first. The factors investigated in this report were as follows: ethanol concentration (0/10%/30%/50%/70%/ 90% v/v), ratio of liquid to solid (5/15/25/45/55 ml/g), ultrasonication duration (10/20/30/40/60/70/80/90/100/ 120 min) and extraction temperature (25/35/45/55/65 C).
Then, the extraction parameters of ultrasonication (i.e. ethanol concentration, ratio of liquid to solid, UAE time and extraction temperature) were optimized by an orthogonal test and further validated by a verification test. The investigated levels of each factor were selected depending on the results of single factor experiments. Multiple sequential extraction (3 · 30 min) of the same ground sample was performed to optimize and validate the number of extraction cycles. Ground sample was sonicated for 30 min and filtered through 0.45 lm microporous membrane. The filtrate was collected and the residue was subsequently extracted twice with the same volume of fresh solvent for the same extraction time. The actual content of individual flavonoid in ground sample was defined as the amount measured by Soxhlet extraction. The amount of individual flavonoid extracted in each ultrasonication cycle was expressed as a percentage of this content (i.e. recovery). In this study, each experiment was performed in triplicate and repeated at least once. 2.4. HPLC analysis Acetonitrile (solvent A) and 1.0% acetic acid (solvent B), deaerated ultrasonically for more than 30 min in advance, were used as the mobile phase with a flow rate of 1.0 ml/ min. The column temperature was 25 C and the sample volume injected was 5 ll. The detector was set at 270 nm. For the elution program, the following proportions of solvent A were used: 0–10 min, 20% solvent A; 10– 40 min, 20–25% solvent A; 40–42 min, 25–50% solvent A; 42 min, 50–20% solvent A. The chromatographic peaks of epimedin A, B, C and icariin were confirmed by comparing their retention time and UV spectra with those of the
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385 30
25
Extraction yield (mg/g)
15
10
25
Extraction yield (mg/g)
epimedin A epimedin B epimedin C icariin
20
5
0
20 epimedin A epimedin C
epimedin B Icariin
15 10 5 0
0
10
30
50
70
90
0
20
Ethanol concentration (%)
40
60
80
100
120
140
Extraction time (min)
Fig. 1. Effect of ethanol concentration on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ratio of liquid to solid: 100 ml/g; UAE time: 90 min; extraction temperature: 25 C; number of extraction cycles: 1.
Fig. 3. Effect of ultrasonic-assisted extraction time on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; ratio of liquid to solid: 30 ml/g; extraction temperature: 25 C; number of extraction cycles: 1.
35 epimedin A epimedin B epimedin C icariin
25 20 15 10
Extraction yield (mg/g)
30
Extraction yield (mg/g)
379
28
epimedin A by UAE
epimedin B by UAE
epimedin C by UAE epimedin A by BE
icariin by UAE epimedin B byBE
epimedin C by BE
icariin by BE
21
14
7 5
0 0 5
15
25
45
55
25
Ratio of liquor to solid (ml/g) Fig. 2. Effect of ratio of liquid to solid on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; UAE time: 90 min; extraction temperature: 25 C; number of extraction cycles: 1.
reference standards. Quantification was carried out by the integration of the peak using external standard method. 2.5. Scanning electron micrographs After extraction, the sample was centrifuged at 3250g for 10 min. The supernatant was discarded and the precipitate was dried. To determine the changes of particle size, the remaining residue was sputtered with gold and then examined by scanning electron microscopy under the high vacuum condition and at an operating voltage of 20 kV. To observe the cell damage, the residue was plunged in liquid nitrogen and then cut with a cold knife. The sectioned particles were sputtered with gold and examined by scanning electron microscopy [28].
35
45
55
65
Extraction temperature (°C) Fig. 4. Effect of extraction temperature on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii. Ethanol concentration: 50%; ratio of liquid to solid: 30 ml/g; UAE time: 30 min; number of extraction cycles: 1; UAE, ultrasonic-assisted extraction; BE, boiling extraction at different temperature.
2.6. Stability test 2.6.1. Stability of ultrasonication method Five replicates of the same ground sample (1.0 g) were processed according to the same optimal UAE protocol. The crude extracts were analyzed by HPLC and the extraction yields of epimedin A, B, C and icariin were calculated. The stability of ultrasonication method was assessed by comparing the results among the five replicates.
2.6.2. Photostability of epimedin A, B, C and icariin in the crude extract The photodegradation of epimedin A, B, C and icariin in the crude extract (aqueous ethanol solution) was carried
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out in quartz cells (optical path 10 mm) where the crude extract was irradiated with a UV lamp for 0–22 h at 25 ± 2 C. In order to avoid overheating of the samples, a cooling fan and an air-conditioner were employed to eliminate the heat from the UV lamp and to control the room temperature, respectively. The distance between the cells and the lamp was 5 cm. Samples were taken at 0, 2, 3, 12, 16, 18, 22 h and the concentrations (ng/ll) of the four flavonoids of each sample were monitored by HPLC. The photodegradation rate of individual flavonoid was calculated according to the following formula: R ¼ ðc0 c1 Þ=c0 100% where R, c0 and c1 were the photodegradation rate (%), the concentration (ng/ll) of individual flavonoid in the initial crude extract prior to UV irradiation and the concentration (ng/ll) of individual flavonoid in the degraded crude extract at different UV radiation time, respectively. 3. Results and discussion 3.1. Optimization of parameters for UAE 3.1.1. Effects of extraction variables Both methanol and ethanol have been successfully used as the solvent for extracting icariin and total flavonoids in Herba Epimedii [23]. However, methanol was regarded as not in compliance with good manufacturing practice (GMP) due to its high toxicity [27]. Ethanol was thus adopted as the solvent for UAE in this study. Fig. 1 shows that the extraction yields of epimedin A, B, C and icariin with aqueous ethanol solution as the solvent are higher than those with water (0% ethanol solution) as the solvent. The results also indicated that the extraction yields of the four flavonoids first increased and then decreased with
the increase of ethanol concentration, with the highest yields in 50% (v/v) ethanol solution. The effects of liquid:solid ratio on the extraction yields of epimedin A, B, C and icariin from Herba Epimedii are shown in Fig. 2. The results demonstrated that the extraction yield of epimedin C rose as liquid:solid ratio increased. When the ratio increased from 15 to 25 ml/g, the yields of epimedin A, B and icariin increased dramatically. In order to obtain more target flavonoids with less solvent, the ratio of liquid to solid should be maintained in the range of 20– 45 ml/g. The influence of UAE time on the extraction of epimedin A, B, C and icariin is shown in Fig. 3. The results indicated that the extraction yields of the four flavonoids increased with the increase of ultrasonication duration. There is an obvious increasing trend for the four flavonoids. For epimedin B, C and icariin, the yield increased rapidly at 10–30 min and then slowly after 30 min. The extraction yield of epimedin A was much lower than the others. But the yield of epimedin A still increased slightly at 10–30 min, and reached the plateau after 40 min. The impacts of extraction temperature on the extraction yields of epimedin A, B, C and icariin are demonstrated in Fig. 4. When the temperature was increased from 25 C to 65 C, the extraction yield of icariin was improved. However, the yield of epimedin B was not significantly affected by extraction temperature. The yields of epimedin A and C rose within the first 45 C, after which the yields declined. The high yields of target flavonoids at elevated temperature may be partially attributed to the increase in the number of cavitation bubbles formed by ultrasound and to the enhanced mass transfer rates [20]. Nevertheless, when temperature rose to a certain degree (i.e. above 45 C), the yields of epimedin A and C decreased. One of the possible reasons for this phenomenon is that epimedin A and C suffered thermodegradation, but the contents of epimedin A,
Table 2 Orthogonal test design and results Test no.
Factor
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a1 a1 a1 a1 a2 a2 a2 a2 a3 a3 a3 a3 a4 a4 a4 a4
(a) Ethanol concentration (%) 30 30 30 30 40 40 40 40 50 50 50 50 60 60 60 60
Extraction yield (mg/g) (b) Ratio of liquid to solid (ml/g) b1 b2 b3 b4 b1 b2 b3 b4 b1 b2 b3 b4 b1 b2 b3 b4
20 25 30 35 20 25 30 35 20 25 30 35 20 25 30 35
(c) Ultrasonic-assisted extraction time (min) c1 c2 c3 c4 c2 c1 c4 c3 c3 c4 c1 c2 c4 c3 c2 c1
20 30 40 50 30 20 50 40 40 50 20 30 50 40 30 20
(d) Extraction temperature (C) d1 d2 d3 d4 d3 d4 d1 d2 d4 d3 d2 d1 d2 d1 d4 d3
40 45 50 55 50 55 40 45 55 50 45 40 45 40 55 50
Epimedin A
Epimedin B
Epimedin C
Icariin
2.2815 3.5943 4.6311 2.7273 4.1845 3.3888 3.8447 3.5100 2.2933 3.0040 4.0025 3.7126 2.5439 2.4010 4.7862 3.2102
22.3098 23.4166 21.3263 22.0101 22.0432 19.7327 19.0011 20.7453 19.5211 20.6122 19.8870 23.9842 18.8712 18.4276 20.9011 21.8392
5.6163 5.8701 7.3727 5.6204 6.5525 5.7325 6.0219 5.7021 6.1033 6.5430 7.4888 6.1138 5.7000 5.6003 6.3020 5.8796
6.1098 8.9707 11.1315 10.5222 11.3012 8.3264 7.0303 10.6213 10.1999 12.0002 10.2096 10.9877 7.8314 6.4726 9.3021 12.6000
H.-F. Zhang et al. / Ultrasonics Sonochemistry 15 (2008) 376–385
B, C and icariin in the crude extract did not declined with the increase of temperature (up to 65 C) without ultrasound (data not shown). To gain further insight into the phenomenon, boiling extraction was modified and performed at different temperature (25/35/45/55/65 C). As shown in Fig. 4, the yields of the four flavonoids obtained by boiling extraction increase with the increase of temperature, which does not indicate thermal instability of the four flavonoids. It is also possible that extremely high temperature decreased the number of cavitation bubbles [29] and weakened the impact of cavity collapse on ground samples, which resulted in the decline of the yields of epimedin A and C. Furthermore, extremely high temperature might lead to some other disadvantages, such as increase of energy cost, acceleration of solvent volatilization and enhancement of impurities extraction. Hence, extraction temperature should not exceed 55 C. Table 3 Extreme difference analysis of orthogonal test Statistical item
Flavonoid
Factor a
b
c
d
381
3.1.2. Results and analysis of orthogonal test In order to obtain an optimal condition under which the extraction yields of epimedin A, B, C and icariin were all high, an orthogonal test was designed to optimize parameters on the basis of the results of single factor experiments (see above). Four factors, ethanol concentration (a), ratio of liquid to solid (b), UAE time (c) and extraction temperature (d), were selected for optimization, each at four levels (Table 2). The extraction yields of the four flavonoids were regarded as investigated indexes. The total evaluation indexes were analyzed by statistical method, i.e. extreme difference analysis and variance analysis. The results of orthogonal test, extreme difference analysis and variance analysis are presented in Tables 2–4, respectively. The analysis of extreme difference revealed that the influential order of the four factors on the extraction yields of epimedin A, B, C and icariin is b > c > d > a, c > a > b > d, b > d > a > c and d > b > a > c, respectively. This order was in agreement with that based on the F values in variance analysis. In addition, according to variance analysis, the contributions of b and c were significant for the extraction yield of epimedin A (P < 0.05). For the extraction yield of epimedin B, all factors except d showed significant influence (P < 0.05), which confirmed the results of single factor experiments. The effects of a, b and d on the yield of icariin were very significant (P < 0.01) and effect of
T1
Epimedin A Epimedin B Epimedin C Icariin
13.2342 89.0628 24.4795 36.7342
11.3032 82.7453 23.9721 35.4423
12.8830 83.7687 24.7172 37.2458
12.2398 83.7227 23.3523 30.6004
T2
Epimedin A Epimedin B Epimedin C Icariin
14.9280 81.5223 24.0090 37.2792
12.3881 82.1891 23.7459 35.7699
16.2776 90.3451 24.8384 40.5617
13.6507 82.9201 24.7610 37.6330
Table 4 Variance analysis of orthogonal test
Epimedin A Epimedin B Epimedin C Icariin
13.0124 84.0045 26.2489 43.3974
17.2645 81.1155 27.1854 37.6735
12.8354 80.0203 24.7784 38.4253
15.0298 85.8209 26.3478 47.0329
a
Epimedin A Epimedin B Epimedin C Icariin
12.9413 80.0391 23.4819 36.2061
13.1601 88.5788 23.3159 44.7312
12.1199 80.4946 23.8853 37.3841
13.1956 82.1650 23.7582 38.3506
Epimedin A Epimedin B Epimedin C Icariin
3.3086 22.2657 6.1199 9.1836
2.8258 20.6863 5.9930 8.8606
3.2208 20.9422 6.1793 9.3115
3.0600 20.9307 5.8381 7.6501
Epimedin A Epimedin B Epimedin C Icariin
3.7320 20.3806 6.0023 9.3198
3.0970 20.5473 5.9365 8.9425
4.0694 22.5863 6.2096 10.1404
3.4127 20.7300 6.1903 9.4083
Epimedin A Epimedin B Epimedin C Icariin
3.2531 21.0011 6.5622 10.8494
4.3161 20.2789 6.7964 9.4184
3.2089 20.0051 6.1946 9.6063
3.7575 21.4552 6.5870 11.7582
Epimedin A Epimedin B Epimedin C Icariin
3.2353 20.0098 5.8705 9.0515
3.2900 22.1447 5.8290 11.1828
3.0300 20.1237 5.9713 9.3460
3.2989 20.5413 5.9396 9.5877
Epimedin A Epimedin B Epimedin C Icariin
0.4967 2.2559 0.6917 1.7979
1.4903 1.8658 0.9674 2.3222
1.0394 2.5812 0.2383 0.8289
0.6975 0.9139 0.7489 4.1081
T3
T4
K1
K2
K3
K4
R
Sourcea Flavonoid
Mean square
Epimedin A 0.664 Epimedin B 11.747 Epimedin C 1.081 Icariin 8.455
3 3 3 3
0.221 3.916 0.360 2.818
2.793 18.260 3.465 105.246
Epimedin A 5.086 Epimedin B 8.417 Epimedin C 2.362 Icariin 14.069
3 3 3 3
1.695 2.806 0.787 4.690
21.393 13.084 7.570 175.141
*
Epimedin A 2.610 Epimedin B 16.992 Epimedin C 0.151 Icariin 1.759
3 3 3 3
0.870 5.664 0.050 0.586
10.976 26.413 0.485 21.902
*
Epimedin A 1.010 Epimedin B 1.864 Epimedin C 1.334 Icariin 33.988
3 3 3 3
0.337 0.621 0.445 11.329
4.248 2.897 4.276 423.096
Error
Epimedin A Epimedin B Epimedin C Icariin
0.238 0.643 0.312 0.080
3 3 3 3
0.079 0.214 0.104 0.027
Total
Epimedin A 9.608 Epimedin B 39.663 Epimedin C 5.240 Icariin 58.351
15 15 15 15
c
d
F
Significanceb
d.f.
b
Sum of squares
*
**
*
**
*
*
**
a a, Ethanol concentration (%); b, ratio of liquid to solid (ml/g); c, UAE time (min); d, extraction temperature (C). b * , Significant (P < 0.05); **, very significant (P < 0.01).
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epimedin A, B, C and icariin in Herba Epimedii by HPLC or capillary zone electrophoresis.
Table 5 Results of verification test Test no.
Factor
Extraction yield (mg/g)
a
b
c
d
Epimedin A
Epimedin B
Epimedin C
Icariin
3.2. Reproducibility of ultrasonication method
17 18 19 20
a2 a1 a3 a3
b3 b4 b3 b4
c2 c2 c2 c2
d3 d3 d3 d3
5.0691 4.2168 4.9032 4.1967
22.8790 28.2213 25.7069 25.6989
6.9089 6.4330 7.5743 6.7223
11.5310 11.8363 12.8466 12.8300
To study the stability of UAE method for epimedin A, B, C and icariin in Herba Epimedii, five ground samples of 1.0 g were processed under the optimized condition, i.e. ethanol concentration of 50%, liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C in three extraction cycles (data not shown). The relative standard deviations (RSDs) for the determined extraction yields of the four flavonoids are found to be less than 3% (n = 5), showing a good reproducibility of UAE method.
c was significant (P < 0.05). However, none of the four factors significantly affected the yield of epimedin C. According to extreme difference analysis, the optimum conditions for extracting epimedin A, B, C and icariin were a2b3c2d3, a1b4c2d3, a3b3c2d3 and a3b4c2d3, respectively. However, all of these conditions were not included in orthogonal test. In order to further validate these optimal conditions, UAE under these conditions was implemented. The results of verification test are listed in Table 5. The highest yields of epimedin C and icariin were obtained under the condition of a3b3c2d3 (Tables 2 and 5). In fact, the yields of epimedin A and B were also high under this condition. So this condition was rationally confirmed to be the most optimum, namely, ethanol concentration of 50% (v/v), liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C.
3.1.3. Validation of number of extraction cycles In general, the extraction amount of constituents increases with the increase of the extraction cycles. In order to investigate the influence of number of extraction cycles on the recoveries of epimedin A, B, C and icariin, ground sample was extracted triplicately (3 · 30 min) under the above optimized condition (a3b3c2d3). The results are listed in Table 6. The recoveries of the four flavonoids slightly increased with the increase of the extraction cycles. After the third extraction cycle, 100% of the four flavonoids were recovered. Therefore, three ultrasonication cycles were performed in sample preparation for the determination of
3.3. Photostability of flavonoids In plants, flavonoids are reported resistant to heat, oxygen, dryness and moderate degrees of acidity but prone to be modified by short wavelength light [30]. The concentrations of epimedin A, B, C and icariin in the crude extract (ethanol solution) decreased as UV irradiation time was extended (data not shown). When epimedin A, B, C and icariin were subjected to UV radiation for 22 h, their photodegradation rates reached 15.16%, 4.69%, 4.73% and 3.78%, respectively. However, no significant photodegradation of the four flavonoids in the crude extract stored in the shade for about 7 days was observed (data not shown). Hence, short wavelength light should be avoided for the extraction and storage of Herba Epimedii samples. 3.4. Comparison of UAE and conventional extraction method The extraction efficiency of UAE was compared with that of other classical methods, Soxhlet extraction and boiling extraction. Table 6 summarizes the results. After extraction for 90 min, the best recoveries (100%) of epimedin A, B, C and icariin were achieved by UAE at the
Table 6 Recoveries of epimedin A, B, C and icariin versus number of extraction cycles in different extraction methods Extraction method
Number of extraction cycles
Ultrasonicassisted extraction
1 2 3
30 60 90
50 C
Boiling extraction
1 2 3 4
90 120 150 180
Soxhlet extraction
1 1
90 240
a b
Extraction time (min)
Extraction temperature
Amount of solvent (ml)a
Recovery (%)b Epimedin A
Epimedin B
Epimedin C
Icariin
30 60 90
93.4 100.0 –
92.6 100.0 –
91.5 99.8 100.0
90.9 99.4 100.0
90 C
50 100 150 200
83.1 95.4 98.7 100.0
96.7 99.1 100.0 –
35.8 82.0 96.0 100.0
69.8 94.4 99.1 100.0
90 ± 2 C
100 100
41.6 100.0
44.8 100.0
37.0 100.0
40.0 100.0
Ground samples of 1.0 g are used in each extraction method. The recovery means the extracted target flavonoid in % related to its content in the studied Herba Epimedii, measured by classical Soxhlet method.
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383
Fig. 5. Scanning electron micrographs of Herba Epimedii leaf samples from different extraction methods at low magnification (100·, A–C) and high magnification (1500·, D–F). A and D, untreated leaf; B and E, after boiling extraction; C and F, after ultrasonication.
extraction temperature of 50 C, which were higher than those by boiling extraction at 90 C and Soxhlet extraction at 90 ± 2 C. After four extraction cycles at 90 C, boiling extraction used a total of 200 ml of 50% ethanol solution as the solvent for 180 min and obtained the same recoveries (100%) of the four flavonoids as UAE with 90 ml of ethanol solution for 90 min at 50 C. After 240 min extraction
at 90 ± 2 C, Soxhlet extraction using 100 ml of 50% ethanol solution as the solvent gave the same efficiency (100%) as UAE with 90 ml of 50% ethanol solution for 90 min at 50 C. Clearly, by reducing the extraction time, the extraction temperature, and the quantity of solvent required, UAE was proven to be a robust method which can offer improved extractability.
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In order to understand the mechanism of UAE and to elucidate each extraction procedure, the leaf samples from different extraction methods (UAE and boiling extraction) were examined by scanning electron microscopy (Fig. 5). Low magnification (100·) micrographs (Fig. 5A–C) indicated that UAE reduced the particle size of Herba Epimedii leaf samples, which increased the number of cells directly exposed to the solvent and thus exposed to ultrasonically induced cavitation. However, boiling extraction did not clearly decrease the particle size. High magnification images at 1500· magnification (Fig. 5D–F) revealed that the surface of the leaf sample was seriously destroyed after UAE, while only some slight ruptures took place on the surface of the sample after boiling extraction. The solvent extraction from the dried leaf materials includes two stages: steeping leaf in solvent and mass transfer of soluble constituents from leaf to solvent [20]. Ultrasound can produce acoustic cavitation and facilitate the swelling and solvatation of the leaves causing enlargement of the pores on the cell surface, which will improve both the solvent penetration into the leaf materials and the release of the cell contents. This mechanism of UAE is different from those of boiling extraction and Soxhlet extraction that depend on a series of permeation and solubilization processes to wash the intracellular analytes out of the matrix [28]. 3.5. Application of UAE method to Epimedium samples from different origins Epimedin A, B, C and icariin of 20 Epimedium species from different origins were extracted under the optimal UAE condition and quantified by HPLC (Table 1). A large variation of the contents of the four flavonoids was found among these Epimedium species from different origins. The contents of epimedin A in the leaves of E. sagittatum, epimedin B in E. brevicornu, epimedin C in E. truncatum and icariin in E. boreali-guizhouense were the highest, respectively. The variation is possibly attributed to several factors, such as Epimedium species, soil and climate of the collection sites. 4. Conclusions The UAE method reported here can offer an effective alternative for simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii. It can be applied to sample preparation for the quality control of Herba Epimedii. Additionally, it might serve as a promising industrial extraction protocol of the four flavonoids. Compared with conventional boiling extraction and Soxhlet extraction, UAE is an easy, efficient and inexpensive method with low toxicity and high reproducibility. The optimal extraction condition is ethanol concentration of 50% (v/v), liquid:solid ratio 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 C and three extraction cycles. In the crude extract, epimedin A, B, C and icariin can be partially degraded with exposure to UV light.
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