虫草素提取

Journal of Chromatography B, 877 (2009) 2135–2141 Contents lists available at ScienceDirect Journal of Chromatography B journa l homepage: www.e lsev ier .com Column ra Cordyce He Ni, Xi Guangdong Pro China Guangzhou 510 a r t i c l Article history: Received 3 Ma Accepted 7 Jun Available onlin Keywords: Cordycepin pr Cordyceps mili Column chrom Macroporous r Solid fermenta Medium waste ainin re di phic for f ater rous r amid er for d to c ion p CCE method has high extraction efficiency, uses a minimum volume of solvent and can be used for both quantitative analysis and large preparations of cordycepin from waste. The preparation method is simple, highly efficient, energy-saving, environmentally friendly, and has been demonstrated to be effective for large preparations of cordycepin from waste with low equipment and operating costs. © 2009 Elsevier B.V. All rights reserved. 1. Introduc Cordycep sinensis, wh multiple ph anti-aging, in cultivatio price of C. s ity product been demo have simila itaris has be through sol 50–60 tons China. The 10-fold exce C. militaris. waste and p and bioactiv 1/1000 dep ∗ Correspon E-mail add 1570-0232/$ – doi:10.1016/j.j tion s militaris belongs to the same genus as Cordyceps ich is a well-known traditional Chinese medicine with armacological functions, such as immunoregulation, antitumor and antimicrobial activities [1–3]. Difficulty n and limited supplies resulted in an increase in the inensis to US$ 12,000 kg−1 in 2006 for an average qual- [2]. In contrast, C. militaris is easily cultivated and has nstrated to be the best substitute for C. sinensis, as both r chemical components and medical functions. C. mil- en widely cultivated for production of fruiting bodies id fermentation with a reported annual production of of dried fruiting bodies in the Guangdong Province of rice-based, solid fermentative medium is required in a ss to the fruiting bodies produced in order to cultivate Large amounts of used solid medium are discarded as ollute the environment. The waste contains mycelium e substances with a cordycepin content of 1/10,000 to ending on the fungus strain and cultivating condition ding author. Tel.: +86 20 8521 2630; fax: +86 20 8521 2630. ress: li haihang@yahoo.com (H.-H. Li). [4–6]. Thus, utilization of the waste media is of both economic and environmental important. Cordycepin, a nucleoside analogue of 3′-deoxyadenosine, was first isolated from C. militaris [7]. It is classified as an anticancer compound [2,8–11] but also has immunoregulatory [3], antibac- terial [12], antifungal [13], antiviral [14,15] and anti-infection [16] properties. It has also been shown to protect neurons from ischemic injury [17] as well having various other functions [1,2,10]. Cordy- cepin incorporates into RNA and causes premature termination of RNA synthesis, making it a valuable tool in the study of gene transcription [2]. Because of its cytotoxicity to terminal deoxynu- cleotidyl transferase positive (TdT+) leukemic cells, cordycepin is under clinical tests as a therapeutic agent for the treatment of TdT- positive acute lymphocytic leukemia (OncoVista, Inc., San Antonio, TX; granted by the US FDA in July, 2007). Cordycepin is also used in various health products worldwide and its cost has recently risen to US$ 4000 g−1 due to increasing demand. Although cordycepin can be chemically synthesized, microbes like C. militaris are still its main source. Several extraction methods have been developed to extract cordycepin from a fermenta- tive solution and from fruiting bodies of C. militaris, including ultrasound- or microwave-assisted extraction, pressurized extrac- tion, soxhlet extraction and reflux extraction. Wang et al. [18] compared thermal reflux and ultrasound-assisted extraction with water or ethanol and found that the thermal refluxing extrac- see front matter © 2009 Elsevier B.V. All rights reserved. chromb.2009.06.009 chromatographic extraction and prepa ps militaris waster medium ao-Hong Zhou, Hai-Hang Li ∗, Wen-Fang Huang vincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South 631, China e i n f o rch 2009 e 2009 e 13 June 2009 oduction taris atographic extraction esin tion a b s t r a c t Large amounts of solid medium cont militaris through solid fermentation, a developed a new column chromatogra this waste and a preparation method imbibed in four times its volume of w were directly separated with macropo precipitation, crystallization, and poly were obtained with 12 volumes of wat through 3 different columns designe following the separation and purificat / locate /chromb tion of cordycepin from Normal University, g cordycepin, used in the industrial production of Cordyceps scarded as waste and contaminate the environment. We have extraction (CCE) method for the extraction of cordycepin from urther separation and purification. Dried waste material was for 6 h, transferred to columns and eluted with water. Eluates esin DM130 columns followed by purification steps, including e column chromatography. Extraction rates of more than 97% a single column and 4 volumes of water for eluates circulated oncentrate cordycepin. Cordycepin (98% pure) was obtained rocesses, with an overall recovery rate of more than 90%. The 2136 H. Ni et al. / J. Chromatogr. B 877 (2009) 2135–2141 tion with water was the best method for extracting cordycepin and polysaccharide from the fruiting bodies of C. militaris; three 90-min extractions with a sample-to-water ratio of 1:10 each is optimal. Song et al. [19] reported that the main factors affecting cordycepin extraction t for ultrasou 20% ethano ods for the C. sinensis a are similar or boiling w was greatly tures. Xia an cordycepin employed a treatment f sample-to-s solid C. mil macerating extraction e energy cons The repo cepin are b column chr tion amoun pure produ cordycepin method req In this highly effic for extracti as a prepara cordycepin. cepin with and in large offers man high extrac mal volume environmen 2. Materia 2.1. Materia Solid wa the cultivat 1 kg of rice 0.2% KH2PO powder, 0.0 before it wa fruiting bo medium (a d. Dried wa 833�m and Cordyce MO, USA). Jackson Inc. DM101 resi dong, China grade and w analytical o 2.2. Ultraso As a con tion of cord ruitin ry. ions es of room t of 1 ent ( ntain . The lumn olid bibe rred uted ns, e naly . cycl oun sed t the next material and the third fraction was used for elut- second column (Fig. 2). Through this cyclic method, the final ing solution was only four times the volume of the dry mate- hile the columns were eluted three times (each with four the volume of solvent) and almost no cordycepin remained columns. atic adsorption and desorption of cordycepin with a orous resin resins were pretreated with the following procedure before e resin was first imbibed in de-ionized water overnight. bsorbed resins were soaked sequentially with 80% ethanol, l l−1 HCl, and 0.1 mol−1 NaOH, each for 4 h or more and d with distilled water after each soaking. gram of pretreated macroporous resin, with surface water ed with a paper towel, was added into 20 ml of extracts justed with HCl or NaOH) in a 150 ml Erlenmeyer flask and ted at room temperature with shaking (120 rpm). At 10 min, , 30 min, 40 min, 60 min, 120 min and 180 min, 0.5 ml of n was taken and centrifuged at 4000 rpm for 5 min. The extraction are the volume ratio of solvent to sample, ime and ethanol concentration. The optimal conditions nd-assisted extraction of cordycepin use 33 ml g−1 of l for 102 min. Yang and Li [20] compared three meth- extraction of cordycepin and other nucleosides from nd C. militaris. They found that extraction efficiencies by liquid extraction using pressurized organic solvent ater extraction. For some nucleosides, the efficiency increased using water extraction at ambient tempera- d Wen [4] tested the microwave-assisted extraction of from solid C. militaris medium. The optimal conditions sample to water ratio of 1:200 with a 50 W microwave or 3 min. Zhong et al. [6] reported that the optimal olvent ratio for soxhlet extraction of cordycepin from itaris medium was 1:16. These methods are based on extraction and are not ideal because of relatively low fficiency, a large volume of extraction solution and high umption. rted separation and purification methods for cordy- ased on ion exchange, activated carbon or silica gel omatography [6,21]. These methods have low adsorp- ts, low recovery rates and are not ideal for generating cts. Chen et al. [21] successfully prepared high-purity using preparative reverse-phase HPLC, although this uires high equipment and production costs. work, we report on the development of a new and ient column chromatographic extraction (CCE) method ng cordycepin from C. militaris waste medium, as well tion method for further separation and purification of Using these methods, we successfully prepared cordy- a purity greater than 98% in both a laboratory setting r scales at costs of less than US$ 100 g−1. This method y advantages over the current technology, including tion efficiencies and recovery rates, the use of mini- s and concentrations of solvents, energy-savings and tal friendliness. ls and methods ls and reagents ste media of C. militaris cultures were generated by ion center in our laboratory. The medium contained in 1.5 l of nutrient solution (1% glucose, 1% peptone, 4, 0.1% MgSO4, 0.1% ammonium citrate, 1% silkworm 01% vitamin B1), and was autoclaved at 120 ◦C for 2 h s used for cultivation of C. militaris. After fermentation, dies of C. militaris were harvested and the culture s shown in Fig. 1) was dried in an oven at 45 ◦C for 2 ste medium was ground and sieved with pore sizes of /or 350�m. pin standard was purchased from Sigma Co. (St. Louis, HPLC-grade solvents were purchased from Burdick & (Muskegon, MI, USA). Macroporous DM130, DM131 and ns were purchased from Shandong Chemical Co. (Shan- ). The ethanol used in all experiments was 95%, food as purchased from local suppliers. Other reagents were r biochemical grade. und-assisted maceration extraction trol for the new extraction method, macerating extrac- ycepin from the waste medium was used under optimal Fig. 1. F laborato condit volum dark at amoun treatm tion co 10 min 2.3. Co As s was im transfe and el fractio were a eluates For high am proces extract ing the extract rial, w times on the 2.4. St macrop All use. Th Fully a 0.1 mo washe One absorb (pH ad incuba 20 min solutio g bodies of C. militaris cultured by solid medium fermentation in our we determined. Dried media were fully imbibed with 4 water (solvent to sample ratio, v/w, pH 6.5) for 6 h in the temperature. Water (60 ◦C) was then added to a final 0 or 20 times the volume and extracted with ultrasound 100 W) in a 60 ◦C water bath for 1 h. The extraction solu- ing the waste medium was centrifuged at 4000 rpm for supernatant was used for HPLC analysis. chromatographic extraction C. militaris waste medium is a water swelling material, it d with 4 volumes of water (pH 6.5) for 6 h before it was to chromatographic columns. The columns were loaded using common procedures. Eluates were collected in ach with a volume equal to four times the sample, and zed by HPLC until no cordycepin was detected in the ic CCE, only the first eluate fraction, which contains a t of cordycepin, was collected as a final extraction and o the separation steps. The second fraction was used to H. Ni et al. / J. Chromatogr. B 877 (2009) 2135–2141 2137 Fig. 2. Cyclic materials. F1, eluates. Each fr Only F1 was co cordycepin The adsorpt calculated. Once ful containing 20 ml of 80% orption solu were calcul 2.5. Column For sepa resins were distilled wa time with 2 the sample analyzed by For purifi crystals we flowed thro After the sa distilled wa evaporated was then cr 2.6. HPLC d An HPLC China) with software w cepin. HPLC (250 mm × v/v); flow ra amount, 10 brane filter other subst standard curves. Peaks for cordycepin and other compounds in the samples were identified by their retention times and co-injection tests with their corresponding standard compounds. ults and discussion lumn chromatographic extraction of cordycepin from C. is waste medium extraction process was separated into two steps. The first volved dissolving the target substances with a minimum vol- f their ideal solvents. As the dried waste medium began to after imbibition, the materials were fully imbibed in open ers with 4 volumes of water for 6 h until the cordycepin ssolved to its maximum amount (data not shown) and was aded onto columns. The second step was to elute the tar- stances from the columns with water. As shown in Fig. 3, raction rates of cordycepin from C. militaris waste medium ultrasound-assisted water maceration were 87.3% and 91.6% and 20 volumes of water, respectively, while the extraction y the 0 vo ethod ion w than optim ion wer mate to d ed t y im . 4A n 35 30 ◦ epin BV h ycep to di ant chromatography extraction of bioactive substances from biological F2 and F3 refer to Fraction 1, 2, and 3, respectively of the collected action contained four times the volume to the dry weight of materials. llected as an extraction solution. contents in the supernatants were determined by HPLC. ion capacity of macroporous resins for cordycepin was ly adsorbed for 3 h in extraction solution, the solution resins was vacuum-filtered. Resins were desorbed in ethanol for 30 min. The cordycepin content in the des- tions was determined by HPLC and the desorption rates ated. separation and purification of cordycepin 3. Res 3.1. Co militar The step in ume o expand contain was di then lo get sub the ext by the with 10 rates b 8 and 1 CCE m extract higher To extract factors of the height indicat be full 6 h (Fig betwee (either cordyc and 30 of cord height signific ration of cordycepin with macroporous resin, pretreated loaded onto columns. The columns were washed with ter until their pH was neutral, and then washed a final bed volumes (BVs) of distilled water (pH 9.0) before s were loaded. Eluates were collected in fractions and HPLC. cation of cordycepin with polyamide resin, cordycepin re dissolved in distilled water. The aqueous solution ugh a prepared polyamide column at a rate of 2 BVs h−1. mple was loaded, the column was eluted with 3 BVs of ter. All eluates were collected together and vacuum- to 1/4 the original volume. The concentrated solution ystallized at 4 ◦C. etermination of cordycepin system (Dalian Elite Analytical Instrument Ltd., Dailian, dual P230 pumps, a UV230+ detector and analytical as used for the detection and analysis of cordy- conditions were as follows: column, YMC-packed C18 4.6 mm, 5�m); mobile phase, methanol:water (20:80, te, 1.0 ml min−1; UV detection at 260 nm; and injection �l. The samples were filtered through a 0.45�m mem- before injection. Quantitative analysis of cordycepin and ances was determined by their peak area based on their efficiency o were increa with 12 volu reached 98% respectively is necessary From the on the extr effects of te Fig. 3. Compa assisted extra represent sequ CCE method were 77.5%, 89.1%, 95.8% and 98.8% for 4, 6, lumes of water, respectively. The extraction rate for the was 11.5% higher than that of the ultrasound-assisted ith, both using 10 volumes of water, and was even 7.2% that of the latter method using 20 volumes of water. ize the conditions for the column chromatographic of cordycepin from C. militaris waste medium, several e tested, including imbibition time, the particle size rial, extraction temperatures, flow rates of eluate and iameter ratio (H/D) of the loaded columns. The results hat 6 h was needed for the dried waste medium to bibed (data not shown). Imbibition times longer than ), the particle size of the material (either ≥833�m or 0�m and 833�m, Fig. 4B) and extraction temperatures C or 60 ◦C, Fig. 4C) did not have significant effects on extraction. The flow rates of eluates (2 BV h−1, 6 BV h−1 −1) had limited but consistent effects on the extraction in, in favor of a low flow rate (2 BV h−1) (Fig. 4D). The ameter ratio (H/D) of material loaded onto columns had effects on the extraction of cordycepin. The extraction f cordycepin increased about 5% when the H/D ratios sed two-fold. Cordycepin was not completely extracted mes of water at a H/D of 2.5:1. The extraction efficiency and 100% when the H/D was increased to 5:1 and 10:1, . These results demonstrate that a minimum H/D of 5:1 for efficient extraction of cordycepin from the material. results above, extraction conditions had limited effects action of cordycepin in the CCE method. We tested the mperatures, flow rates of eluates and H/D of the column rison of column chromatographic extraction (A) and ultrasound- ction of cordycepin (B) from C. militaris waster medium. �, , � ential extraction, represents one-step extraction. 2138 H. Ni et al. / J. Chromatogr. B 877 (2009) 2135–2141 Fig. 4. Effects aste m thickness: 833 (white (white). (E) He riatio on extractio militaris wa nificant effe cepin (data a componen Based on chromatogr lows. Dried 833�m, im ature (30 ◦C an H/D ratio rate of 2 BV processing. that more t charides (da Our res method ha obtained w reported re with less s method can the fruiting fruiting bod ing solvent was tested plant mater green tea a acuminate completely by HPLC) fr of 30–60% green tea o from C. acum method wa extraction a ical materia 3.2. Cyclic c militaris wa As show from the ch racti conte seco f ext circu umn . Thr te o , a va or on singl atogr vent cy. cycl ed s ns wi aste xtrac ere actio of column chromatographic conditions on cordycepin extraction from C. militaris w �m (black), and 350–833�m (white). (C) Temperatures: 30 ◦C (black) and 60 ◦C ight to diameter ratio (H/D) of extracting material in columns: 2.5:1 (black), 5:1 (st n efficiency of adenosine from the same material of C. ster medium. These three factors showed more and sig- cts on the extraction of adenosine than on that of cordy- not shown). The main reason may be that cordycepin is t outside of cells, while adenosine is within cells. these results, the optimal conditions for the column aphic extraction of cordycepin were determined as fol- waste medium from C. militaris was ground to sizes of bibed with 4 volumes of water for 6 h at room temper- ), and then loaded onto chromatographic columns with of 10:1. The columns were eluted with water at a flow h−1. The eluates were collected for analysis and further Repeated experiments under these conditions showed han 97% of cordycepin and more than 96% of polysac- ta not shown) were extracted from the waste medium. ults indicate that this chromatographic extraction s a higher extraction efficiency compared to those ith ultrasound-assisted water extraction or to previously sults [4,6]; this increased efficiency is accomplished olvent and simple equipment and conditions. This also be used for the extraction of active substances from three f cepin in the ume o which ent col (Fig. 2) tion ra 97.24% times) cyclic chrom the sol efficien The amplifi colum dried w scale e tests w two fr bodies of C. militaris (data not shown), in which dried ies can be loaded directly onto columns with extract- (water) without a separate imbibition step. This method for the extraction of bioactive substances from several ials, including polyphenols, caffeine and theanine from nd camptothecin from the dried leaves of Camptotheca Decne. With ideal solvents, target substances can be extracted (until substances in eluates cannot detected om their biological materials with 12 (4 × 3) volumes ethanol for polyphenols, caffeine and theanine from r 20 (5 × 4) volumes of 60% ethanol for camptothecin inate Decne (unpublished data of our laboratory). The s demonstrated to be particularly useful for the accurate nd quantitative determination of substances in biolog- ls. hromatograph