Extraction Parameters And Drying Methods Of Cistanche Deserticola
May 24, 2024
Cistanche is the Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schenk) Wight dry fleshy stem with scaly leaves of the Orobanchiaceae plant Cistanche deserticola Y. C. Ma [1]. The main active ingredient of Cistanche deserticola is phenylethanol glycosides [2-4]. Pharmacological studies have shown that it has the functions of aphrodisiac, improving immunity, and enhancing memory [4-9].
In recent years, response surface methodology has been widely used in the extraction of traditional Chinese medicine [10-14]. The response surface method has the inherent advantages of high detection accuracy, less number of experiments required, accurate prediction of function models, and ability to reflect the relationship between factors and predicted values [15-17]. Therefore, this experiment uses the comprehensive score of the content of the five phenylethanol glycosides as the response value, and uses Design-Expert 8.0.6.1 software to design the experiment, optimize the extraction process of Cistanche deserticola, and provide information for the identification and establishment of quality standards of Cistanche deserticola.
Currently, the index components echinaceaside and verbascoside are hot research topics in the extraction and processing technology of Cistanche deserticola. The 2015 edition of the Chinese Pharmacopoeia stipulates that the processing method of Cistanche deserticola is "remove impurities, wash, moisten, cut into thick slices, and dry." [1], there are no clear regulations on the drying method. Therefore, this experiment used eight different drying methods, including sun drying, shade drying, oven drying (40, 60, 80, 100, 120 ℃), and freeze drying, to process Cistanche deserticola [18], which provided theoretical basis for the drying process of Cistanche deserticola.
On guidance.
CISTANCHE EXTRACT WITH 30% ECHINCAOSIDE
1 Materials and instruments
Fresh Cistanche deserticola was harvested in October 2018. The place of origin is Mitan Township, Minqin County, Baiyin, Gansu Province. Associate Professor Guo Yehong of Gansu Agricultural University identified it as Cistanche deserticola Y. C. Ma, a fleshy stem with scale leaves. Reference substances echinaceaside (batch number 82854-37-3, mass fraction ≥98%), cistancheaside A (batch number 93236-42-1, mass fraction ≥95%), verbascoside (batch number 61276-17-3, mass Fraction ≥ 98%), isomurabasin (batch number 61303-13-7, mass fraction ≥ 98%), 2′-acetyl verbascoside (batch number 94492-24-7, mass fraction ≥ 95%) were purchased from Chengdu Pu. Feide Biotechnology Co., Ltd.; formic acid and methanol are of chromatography grade, Merck Company of Germany; water is purified water, and other reagents are of analytical grade.
Waters Acquity liquid chromatograph, PDA detector, Empower chromatography workstation and quaternary solvent management system; KQ-100E ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.
Company; AL104 analytical balance, Mettler-Toledo Instruments Co., Ltd.; LyoQuest-85 freeze dryer, Telstar, Spain.
2 Methods and results
2.1 Preparation of reference solution
Precisely weigh the appropriate amounts of echinaceaside, cistancheoside A, verbascoside, isorbascoside, and 2'-acetyl verbascoside reference standards, and add 70% methanol to make the mass concentrations 1.04, 0.61, 0.63, 0.39, and 0.48 mg/ mL of mixed reference solution.
2.2 Chromatographic conditions of Cistanche deserticola
Waters Symmetry C18 (250 mm×4.6 mm, 5 μm); column temperature 30 °C, volume flow 1.0 mL/min; mobile phase is methanol-0.1% formic acid aqueous solution, elution gradient: 0~12 min, 29%~34% Methanol; 12～40 min, 34%～40% methanol; 30～35 min, 40%～35% methanol; 35～40 min, 30% methanol;
1-echinacoside 2-cistanche A 3-verbascoside 4-isoacteoside 5-2′-acetylacteoside
Fig. 1 HPLC of mixed reference (A) and C. deserticola sample (B)
2.3 Preparation of test solution
Precisely weigh 1 g of dried Cistanche deserticola powder into an Erlenmeyer flask (passed through a 65-mesh sieve), add 50 mL of 50% methanol, weigh the mass, ultrasonic at 40 kHz at 150 W, cool to room temperature, and make up the mass to get it.
2.4 Investigation of linear relationship
Take 0.1, 0.5, 1, 2, 4, and 8 mL of the above mixed reference solution into a 10 mL volumetric flask, and add 70% methanol to the mark. Precisely draw 10 μL and measure according to the chromatographic conditions of "2.2". The mass concentration is the abscissa (X) and the peak area is the ordinate (Y). Perform linear regression and the regression equations are: Echinacea Y=128 049 X -830 189, r=0.999 5, linear range 10.4~832.0 μg/mL; Cistancheside AY=87 980 -447 963, r＝0.999 8, linear range 6.3～504.0 μg/mL; Isomurabasin Y＝92 831 Glycoside Y=79 207 X-304 428, r=0.999 4, linear range 4.8~384.0 μg/mL.
2.5 Methodological review
2.5.1 Precision inspection
Take 20 μL of the mixed reference solution in item "2.1" and inject 6 times continuously to measure the peak areas of echinaceaside, cistancheaside A, verbascoside, isorebascoside, and 2'-acetylverbascoside. The RSDs are 0.23% respectively. , 0.71%, 0.59%, 0.41%, 0.29%, indicating that the instrument has good precision.
2.5.2 Repeatability test
Take 6 portions of the same Cistanche deserticola sample, prepare the test solution according to item "2.3", inject the sample under the chromatographic conditions of item "2.2", and measure the peak area. Results The average mass fractions of echinaceaside, cistancheoside A, verbascoside, isorebascoside, and 2′-acetyl verbascoside were 1.83%, 0.21%, 2.34%, 0.13%, and 0.68% respectively, and the RSDs were 1.32 respectively. %, 0.83%, 1.24%, 2.19%,
0.78%, indicating that the method has good repeatability.
2.5.3 Stability test
Determine the stability of the same test solution (prepared according to "2.3") at 0, 2, 4, 8, 12, 24, and 48 hours to obtain echinaceaside, cistancheoside A, verbascoside, and isolebascoside. , 2′-acetyl verbascoside peak area, RSD were 0.58%, 0.21%, 1.58%, 1.46%, 2.19% respectively, indicating that the test solution was stable within 48 h.
2.5.4 Sample recovery test
Take 1 g of 6 Cistanche deserticola samples with known content, and accurately add a certain amount of mixed reference solution (Echinaceaside 1.06 mg/mL, Cistancheoside A 0.86 mg/mL, Verbascoside 2.17 mg/mL, Isolebascoside 0.79 mg /mL, 2′-acetyl verbascoside 0.91 mg/mL), add 70% methanol to make up to 50 mL, and inject samples according to the chromatographic conditions of "2.2". The average recoveries measured are 101.52%, 99.21%, and 102.62% respectively. , 98.62%, 97.36%, RSD are 1.64%, 1.95%, 0.87%, 1.59%, 1.13% respectively, and the sample recovery rate is good.
Cistanche is a well-known tonic herb that can nourish and protect the kidneys. In traditional Chinese medicine theory, Cistanche is the best herb for kidneys.Cistanche is rich in echinacoside, acteoside, and flavonoids.These effective ingredients in Cistanche can reduce kidney cell apoptosis and increase kidney cell proliferation. Therefore, Cistanche is a natural kidney supplement.
