Chemical Properties Of A Mannoglucan From Cistanche Deserticola

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

1. Materials

Oral pastes for horses from two different manufacturers were used containing 10 mg/g (A) or 20 mg/g (B) of ivermectin, respectively. The quantitative assay for ivermectin was performed and the content was 104.4– 107.7% of the declared concentration.

2. Dissolution test

The test was performed with a Ph. Eur. paddle apparatus (Pharma Test, Heinberg, Germany), at agitations of 75 rpm or 50 rpm, at 37 -0.5 C. Sodium lauryl sulfate (BDH Chemicals, Poole, England) 0.5% (w/v) aqueous solution was used as a dissolution medium (900 ml). The paste (4 g of A or 2 g B) was placed into the vessel with a syringe with a cannula (A) or dropping the sample at the surface of the dissolution medium (B). After the time intervals (15, 30, 45, 60 min, and 2 h), a sample of the dissolution medium was withdrawn (2 ml), filtered through a glass filter, and analyzed.

3. HPLC analysis

The determination of ivermectin was performed with a modified HPLC method as described in Ph. Eur. The analyzed solutions were diluted with methanol (1: 10). The HPLC system consisted of an octadecyl column (250 -4 mm; 5 mm; Lichrospher RP-18; Merck), an integrator D-12500 A, a detector UV-Vis L-4250, pump L-6200A (Merck-Hitachi, Darmstadt, Germany). A mixture of acetonitrile: methanol: water (64: 24: 16) was used as the mobile phase with a flow rate of 1.9 ml/min. The volume of the injected sample was 20 ml and detection was performed at 254 nm.

4. Solubility studies

An access of the substance was shaken in tightly closed glass flasks with a suitable solvent (Table 1) for 20 h at 37 0.1 C. The suspensions were filtered and the filtrate, after dilution in methanol, was analyzed by HPLC.

5. Stability in acidic solution

Solutions of ivermectin in HCl 0.1 M were stored in tightly closed glass vials at 20 C and 37 C. Chromatograms of the solutions were recorded at t ¼ 0 and after 1, 2 and 6 h. The area of the ivermectin (retention time 10.5 min) and additional peaks were calculated.

References

FDA Guidance for industry: SUPAC-SS nonsterile semisolid dosage forms. Scale-up and postapproval changes: chemistry, manufacturing, and controls; in vitro release testing and in vivo bioequivalence documentation. May 1997. FIP guidelines for dissolution testing of solid oral products (1997) Dissolute. Technol. 4: 5–14. Siewert M, Dressman J, Brown C, Shah VP (2003) FIP/AAPS guidelines to dissolution/in vitro release testing of novel/special dosage forms, AAPS PharmSciTech, 4 (1) article 7 Modern Research Center for Traditional Chinese Medicine, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China

Xiang mei Wu, Pengfei Tu Received February 13, 2004, accepted April 4, 2004, Prof. Dr. Pengfei Tu, Modern Research Center for Traditional Chinese Medicine, School of Pharmaceutical Sciences, Peking University Health Science Center, No. 38, Xueyuan Road, Haidian District, Beijing 100083, China Pengfeitu@bjmu.edu.cn Pharmazie 59: 815–816 (2004)

A new mannoglucan from Cistanche deserticola from China is characterized. The compound is responsible for a mild stimulation of mitogen-induced T and B lymphocyte proliferation.

Cistanche deserticola Y. C. Ma is a holoparasite growing on the roots of the dicotyledonous plant Haloxylon ammodendron Bunge which is widely distributed in the northwest of China. As it is an important tonic in oriental medicine, many active components and pharmacological activities have been claimed (Karasawa et al. 1986; Yoshizawa et al. 1990). These properties might be associated with some of the polysaccharide components of Cistanche which have not yet been investigated. Mitogenic pectic polysaccharides and neutral polysaccharides have been isolated and characterized from Cistanche deserticola from Mongolia (Radna et al. 1996; Anna et al. 1997; Bozena et al. 1999). Here we report the chemical properties of a new mannoglucan from Cistanche deserticola from China, which is responsible for mild stimulation of mitogen-induced T and B lymphocyte proliferation. After the stems of Cistanche deserticola Y. C. Ma were extracted with 95% ethanol, cold distilled water was used to extract the residue. The cold water extract was precipitated with EtOH, the protein was removed and dialyzed, obtaining the crude polysaccharide CCDP. The crude polysaccharide was fractionated on a DEAE column and a Sephadex G-150 column to afford a protein-free polysaccharide (CDP-6), which comprised glucose (89.2%) and mannose (10.4%), and of which the average Mr was estimated to be 6.8 -104. The polysaccharide CDP-6 showed [a 20 D 42 (c 1, H2O). The absolute configurations of the sugars were determined by GC of TMS (– )-2-butyl glycosides. CDP-6 was methylated (Needs et al. 1993), hydrolyzed, converted to the alditol acetates, and analyzed by GC and GC-MS (Table). This result together with molar ratio data from methylation analysis suggested that CDP-6 has a predominantly 1,6-linked Glcp and a backbone 1,6- linked to a smaller extent branched at O-3 of mannose. The methylation analysis of two polymers (CDP-6-1, CDP-6–2) from partial hydrolysis confirmed that CDP-6 has a backbone consisting of 1,6-linked glucosyl residues and 1,6-linked mannosyl residues, which are substituted at O-3 of mannose by branches consisting of the terminal, 1,6- linked glucosyl residues, and 1,3,6-linked mannosyl residues. According to molar ratios and the literature (Bozena et al. 1999; Bacon et al. 1996), the 13C NMR spectrum of CDP-6 showed signals due to anomeric carbons of the a- d-Glcp (C-1 at 98.4 ppm), bd-Map (C-1 at 104.3–104.9 ppm) and a-d-Map (C-1 at 100.0 ppm). The 1 H NMR spectrum of CDP-6-2 in D2O showed proton signals corresponding to the a-d-Glcp (H-1 at 4.94–4.95 ppm) and a- d-Map (H-1 at 4.96–5.00 ppm). The 13C NMR spectrum of CDP-6-2 in D2O showed proton signals corresponding to the a-D-Glcp (C-1 at 98.6 ppm) and a-d-Map (C-1 at 98.4ppm). Mannoglucans are reported to have a 1,4-linked glucopyrannan and 1,4-linked mannopyrannan backbone (Radjabi Nassab et al. 1984; Tanabe et al. 2000). CDP-6 has some different structural features by its a-1,6-linked Glcp and b-1,6-linked Map backbone, and it's substituted by mannoglucan branches (Fig.). In order to investigate the use of Cistanche deserticola in folk medicine, the effect of CDP-6 on the T and B cell systems was evaluated. It was found to mildly stimulate mitogen-induced T and B lymphocyte proliferation.

Experimental

1. General procedures

Optical rotation was measured with a W22-1S automatic polarimeter. IR spectra were determined on a Perkin-Elmer 599B spectrometer. GC analysis was performed on an Agilent HP6890N instrument, equipped with an FID detector. GC-MS was performed on a Finnigan Trace GC-MS instrument. Homogeneity and Mr of CDP-6 were estimated from the calibration curve of the elution volume of standard dextrans, which was performed on

an Agilent 1100 series apparatus with a Shodex KS-805 column.1 H NMR and 13C NMR spectra were recorded with an INOVA-500 instrument. Protein content was analyzed by the method of Bradford (Andre et al. 1975). Monosaccharides were analyzed as to their alditol acetate after they had been hydrolyzed (2 M TFA, 2 h, 110 C), reduced and acetated. The hydrolysates were analyzed using TLC on a silica gel plate containing 5% sodium dihydrogen phosphate in BuOH-EtOAc-isopropyl alcohol-HOAC––H2Opyridine (3.5: 10: 6: 3.5 : 3 : 3) and sugars were detected by spraying with orcinol-H2SO4. The resulting alditol acetates were analyzed by GLC using an HP-5 capillary column (30 m 0.32 mm). CDP-6 was methylated by the Ciucanu method, followed by hydrolysis, and was analyzed as partially methylated alditol acetates by GC-MS. After partial hydrolysis, the products were dialyzed and separated, obtaining two polymers, CDP-6-1 and CDP-6–2.

2. Plant material

The plant was collected from Inner Mongolia Province in China in August 2001 and was identified by Hu-Biao Chen, Department of Natural Medicine, School of Pharmaceutical Sciences, Peking University Health Science Center. A voucher specimen [E-1-(9)] of this plant was deposited at the School of Pharmaceutical Sciences, Peking University. 3. Extraction, isolation, and purification of polysaccharide CDP-6 The stem of Cistanche deserticola Y. C. Ma (1500 g) were extracted with 95% ethanol (6 L) under reflux. The dried ethanol-insoluble residue was macerated three times with cold distilled water per 12 h. The cold water extract was filtered and the filtrate centrifuged. After precipitation with four volumes of EtOH (stirring and standing for 24 h at 4 C), the precipitate product (35.5 g) was obtained. The protein in the precipitate was removed by Savage’s method. After dissolution in distilled water, the residue (25.5 g) was dialyzed and lyophilized (yield: 10.6 g). The crude polysaccharide (8 g) was dissolved in distilled water (100 mL, 8%w/v) and separated using a DEAE column (70 5 cm, the gradient of 0–2 M NaCl, per 1.5 L). A water eluted fraction (600 mg) was obtained. A sample (250 mg) was purified by column gel-permeation chromatography on a Sephadex G- 150 columns (4 - 80 cm, 1600 mL, 5 mL per fraction). Fractions of CDP-6 (1500–1600 mL) were pooled, dialyzed, and freeze-dried. After being desalted by Sephadex G-25 (60 1 cm). CDP-6 (110 mg) was obtained and determined as a single peak by HPSGC.

Acknowledgment: The author thanks Dr. Baomei Shao and Prof. Xiaoming Gao, School of Basic Medical Sciences, Peking University Health Science Center, for measuring the immunomodulating activity of CDP-6.