This invention relates to new chemical compounds isolated and purified from herbal medicines. Particularly, it relates to two previously unknown compounds which are glycosides of psoralen and isopsoralen, respectively, and relates to a method of using the two compounds as therapeutics agents and as a means for quality control and evaluation of the herbal medicine Fructus Psoraleae.
Fructus Psoraleae, the dried fruits of Psoralea corylifolia L., is one of the most popular traditional Chinese medicines and is officially listed in the Chinese Pharmacopoeia. This herbal medicine has been used for the treatment of “enuresis, pollakiuria, weak kidney, and pain and cold in the waist and knees,” a group of diseases defined under the Traditional Chinese Medicine (TCM). It has always attracted research interest and, in the year of 2005 alone, over ten articles was published, all focusing on its quality analyses[1-3], photochemistry[4-8], and bioactivities[9-16].
Previous photochemistry revealed that Fructus Psoraleae contains psoralen, isopsoralen, psoralidin, corylifolin, isobavachalcone, corylin, and backuchiol. Among them, two major coumarins, psoralen and isopsoralen, are commonly used as quality markers, or in other words, the coumarins are regarded as the main active ingredients of the herbal medicine. All previously reported quality control methods for this important herbal medicine were based on the coumarins. It was unknown prior to the present invention that any benzofuran glycosides would exist in the herb Fructus Psoraleae.
As an object of the present invention, there is provided two novel chemical compounds and their functional derivatives. The two parent compounds were previously unknown and were newly isolated and purified from the herbal medicine Fructus Psoraleae. They are benzofuran glycosides of formula (1) and (2), respectively. Based on the teaching of the present invention, people of ordinarily skill in the art would understand that the benzofuran glycosides are one of the main active ingredients contributing to the herbal medicine's therapeutic effects. The present invention also provides a method for isolating and purifying the benzofuran glycosides from herbals.
As another object of the invention, there is provided a method for quality control and evaluation of the herbal medicine Fructus Psoraleae using the two compounds as chemical markers, alone or combined with other previous known chemical markers. This quality evaluation method yields significantly better repeatability and consistency than any previously known methods.
Isolation of the New Compounds
Equipment Setup: ESI-MS was recorded on a VG Auto Spec-3000 spectrometer. ID-and 2D-NMR spectra were run on a Brucker AM-400 and a DRX-500 instrument with TMS as internal standard, respectively. The HPLC-ESI-MS analyses were carried out using an Agilent 1100 series which chromatographic system consisted of a binary pump, photodiode array detector (DAD), coupled to an Agilent MSD-Trap SL through a Brucker atmospheric pressure chemical ionization (APCI) interface. The APCI-MS spectra were acquired over a range of m/z 100-900 in positive ion mode. The temperature of dry gas and APCI were set at 350 □and 400 □, respectively. The nebulizer gas pressure was 50 psi and the dry gas flow rate was 51/min. The HPLC system was directly connected to the MS without stream splitting. The injection volume of sample was 10 μl. The Agilent 1100 series HPLC system equipped with Zorbax® XDB-C8 analytical column (4.6×150 mm, 5 μm, Agilent Technologies, U.S.A.), a C18 guard column (4.6×12.5 mm, 5 μm, Agilent Technologies, U.S.A.), a DAD, and an Alltech ELSD 2000 detector (USA) was setup for the analysis. The analysis was performed at 20 ° C. during the whole process. The mobile phase was a mixture of methanol and 0.1% acetic acid at a flow rate of 1.0 ml/min. Linear gradient elution from 10% to 88% methanol (v/v) in 40 min was applied. Herbal Material: The dried fruits of P. corylifolia were purchased in Shenzhen, China, and authenticated by Dr. C. F. Qiao, Chinese Medicine Laboratory, Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong, China. A specimen (No CMED-0178-26) was deposited at the Chinese Medicine Laboratory, HKJCICM.
Extraction and Isolation: The herbal material (1 kg) was processed into powder, which was then refluxed twice with 5 L EtOAc. The residue was then refluxed twice each with 5 L MeOH. The MeOH extracts were combined. The combined extract was filtered and evaporated to dryness by rotary evaporation below 60° C. under reduced pressure. The dried residue was suspended in water. The water solution was loaded on a Diaion HP-20 macroporous resin column, eluting with water. The glycoside fraction was collected based on the absorption peak as detected by a built-in detector of the HPLC equipment. Separation of the target fraction on an ODS column eluting with 5% MeOH was repeated until yielding two compounds, which were subsequently identified as benzofuran glycosides, whose structure are defined by formula (I) and (II), respectively. These two compounds were further purified on a Sephadex LH-20 column eluted with MeOH and subject to MS and NMR analyses for structural identification.
Identification of the New Compounds
By using the foregoing disclosed method, the two new compounds were obtained and shown as major peaks in the HPLC chromatogram (never be reported before). Interestingly, when the herbal material was extracted with boiling water, these two compounds were not present in the extract, even if they have a high polarity close to sugar.
The analyses with high performance liquid chromatography electrospray ionization mass spectroscopy (HPLC-ESI-MS) showed that the two compounds had the same MS spectra, in which three major ion peaks were displayed clearly. As shown in
Nine other compounds were also isolated from the herbal materials, which were known. By comparing with the reported data [3,5], they were identified to be psoralen, isopsoralen, psoralidin, corylifolin, corylin, corylifolinin, isobavachalcone, corylifol A, and bakuchiol.
Obtaining benzofuran glycosides from herbs proved to be challenging work. In a previously reported phytochemical study on the seeds of Psoralea plicata [19], several similar benzofuran glycosides were reported. These glycosides, however, were obtained as acetates. Acetylization had to be carried out to improve the separation by changing the chemical and chromatographic properties of these glycosides. In other words, those glycosides obtained did not have free hydroxyl groups. In the present invention, the glycosides were first isolated by repeated column chromatography (CC) on ODS. Although the HPLC analyses (equipped with UV detector) showed only one peak, they were proved not pure by NMR analysis. The impurity was revealed by evaporative light scattering detector (ELSD), and was finally removed by CC on Sephadex LH-20 eluting with MeOH.
Therefore, the present invention has not only provided two new compounds but removed the uncertainty regarding the nature of benzofuran glycosides reported previously. Based on the biosynthesis relationship between these glycosides and their aglycone derivatives (psoralen and isopsoralen), it can now reasonably conclude that the cinnamic acid group is cis-form. The trans-form of these reported glycosides might be derived from the related acetylization. This conclusion is entirely consistent with the discovery of the cis-isomeric glycosides (compounds 1 and 2) of the present invention.
Effects of Extracting Methods on the Yielding of Glycosides:
According to the present invention, the yielding of glycoside compounds 1 and 2 varies according to the particular method employed for extraction. Specifically, if the herbal material was extracted using water or a water/alcohol mixture as the solvent, compounds 1 and 2 were converted to compounds 3 and 4 to varying degrees, respectively. While not intended to be bound to the theory, applicants believe that this conversion may take place according to the scheme shown in
Use of the New Compounds as Chemical Markers for Quality Assessment
Reagents and Materials: Twenty-three batches of raw herbal material of Fructus Psoraleae were collected from different regions of China and they were authenticated as genuine P. corylifolia according to their morphological characteristics. The reference chemical standards of psoralen (batch No. 110739-200309) and isopsoralen (batch No. 110738-200410) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). Psoralenoside and isopsoralenoside were isolated and purified as described in the foregoing. The purities of the chemical references were determined to be higher than 98% by HPLC analysis. HPLC grade methanol and acetic acid were purchased from International Laboratory (Nevada, U.S.A.). HPLC grade water was prepared by Millipore Milli-Q SP water purification system.
Equipment Setup: The Agilent 1100 series HPLC system equipped with a Zorbax® XDB-C8 analytical column (4.6×150 mm, 5 μm, Agilent Technologies, U.S.A.), a C18 guard column (4.6×12.5 mm, 5 μm, Agilent Technologies, U.S.A.) and a photodiode array detector (DAD) was set up for the analysis. The analysis was performed at 20 ° C. during the whole process. The mobile phase was a mixture of methanol and water (containing 0.1% acetic acid) at a flow rate of 1.0 ml/min. Linear gradient elution from 25% to 60% methanol (v/v) in 30 min was applied. The detection wavelength was set at 246 nm.
Standard solutions: The stock solutions were prepared in methanol at the concentrations of 0.636 mg/ml (psoralen), 0.588 mg/ml (isopsoralen), 0.611 mg/ml (psoralenoside) and 0.587 mg/ml (isopsoralenoside). The stock solutions were diluted and mixed in 5 ml volumetric flasks to yield a series of standard solutions with different concentrations for the validation of linearity.
Sample Solutions: Samples were pulverized to power, which was screened through 355 μm sieves. The fine powder (1 g) was accurately weighed, and mixed with 50 ml methanol in a flask (100 ml). The flask was weighed again. Then, the powder was refluxed in methanol for 4 hours. After cooling, methanol was added to make up to the initial weight. The supernatant fluid was filtered through a syringe filter (0.45 μm). 5 μl of this solution was injected into the HPLC system for analysis.
The Experimental Data as Basis for Herbal Quality Assessment: Table 2 lists the 23 samples of Fructus Psoraleae collected from different regions of China that used in the example. For each sample, the HPLC chromatogram was generated at wavelengths of 246 nm (shown in
Validation of the Quantitative Analysis: The method for quantitative analysis of four constituents in Fructus psoraleae was validated in terms of linearity, recovery and repeatability. Linearity was examined with standard solutions. The linear relationships between the injection quantities (μg, x-asis) and peak area ratio (y-axis) were expressed by the equations listed in Table 3. The correlation coefficients ranged from 0.9997 to 0.9999, and the calibration curves were straight lines. Quintuplicate samples were spiked with known amounts of these four compounds and then extracted in order to study the recoveries. The average recoveries ranged from 96.47 to 100.73%, and the ranges of relative standard deviations (RSD) were from 2.19 to 3.57%. Five individual samples from the same batch were extracted and processed in accordance with sample preparation procedures for quantitative analysis. The RSD of the determination results of these four constituents ranged from 1.98 to 3.03%. It indicated that the repeatability is suitable for quantitative analysis. The validation results (calibration equation, recovery and repeatability) are listed in Table 3.
Contents of the Four Compounds in Tested Samples:
Manufacturing Pharmaceutical Compositions Containing Psoralenoside and Isopsoralenoside:
As shown in the foregoing, psoralenoside and isopsoralenoside are major compounds in Fructus Psoraleae, a popular herbal material used under the TCM for thousands of years and it is contemplated, as people with ordinary skill in the art would do, that the two newly discovered compounds are ingredients contributing to the herbal's therapeutic effects and that it is desirable at times to prepare pharmaceutical composition from substantially pure psoralenoside and/or isopsoralenoside, which may isolated from herbals or through total or semi chemical synthesis. As it is the status of the art in the pharmaceutical industry, once substantially pure preparations of a compound are obtained, various pharmaceutical compositions or formulations can be prepared from the substantially pure compound using conventional processes or future developed processes in the industry. Specific processes of making pharmaceutical formulations and dosage forms (including, but not limited to, tablet, capsule, injection, syrup) from chemical compounds are not part of the invention and people of ordinary skill in the art of the pharmaceutical industry are capable of applying one or more processes established in the industry to the practice of the present invention. Alternatively, people of ordinary skill in the art may modify the existing conventional processes to better suit the compounds of the present invention. For example, the patent or patent application databases provided at USPTO official website contain rich resources concerning making pharmaceutical formulations and products from effective chemical compounds. Another useful source of information is Handbook of Pharmaceutical Manufacturing Formulations, edited by Sarfaraz K. Niazi and sold by Culinary & Hospitality Industry Publications Services.
It is further contemplated that the novel compounds psoralenoside and isopsoralenoside may be modified in various ways which are known in the art. Therefore the compounds of the present invention encompass all the compounds having the backbone structure defined by formula (1) or (2).
The term “pharmaceutical carrier” means an ingredient contained in a drug formulation that is not a medicinally active constituent. The term “an effective amount” refers to the amount that is sufficient to elicit a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment. A person skilled in the art may determine an effective amount under a particular situation.
A “pharmaceutically acceptable carrier” is determined in part by the particular composition being administered and in part by the particular method used to administer the composition. A wide variety of conventional carrier may be suitable for pharmaceutical compositions of the present invention and can be selected by people with ordinary skill in the art.
While there have been described and pointed out fundamental novel features of the invention as applied to a preferred embodiment thereof, it will be understood that various omissions and substitutions and changes, in the form and details of the embodiments illustrated, may be made by those skilled in the art without departing from the spirit of the invention. The invention is not limited by the embodiments described above which are presented as examples only but can be modified in various ways within the scope of protection defined by the appended patent claims.
This application claims priority to U.S. Provisional Application No. 60/752,960, filed Dec. 23, 2005, the contents of which are hereby incorporated by reference.
Number | Date | Country | |
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60752960 | Dec 2005 | US |