Abstract:
The present invention disclosed a compound, proanthocyanidin A1 (PA1), which is isolated from  Lasia spinosa  (L.) Thwait, for its effects in shortening of clolon length, reducing colonic tissue damage, suppressing colonic myeloperoxidase activity with colitis. The said compound can be used in treating inflammatory diseases and anti-cancer leading compound.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/183,728 filed Jun. 23, 2015; 62/183,729 filed Jun. 23, 2015 and 62/183,726 filed Jun. 23, 2015; the disclosures of which are incorporated herein by reference in their entirety. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention relates to a therapeutic effect of proanthocyanidin Al (PA1). More particularly, it relates to a compound that is naturally occurring in  Lasia spinosa  (L.) Thwait and its effects of treating an inflammatory disease or as an anti-cancer leading compound. 
       BACKGROUND OF INVENTION 
       [0003]      Lasia spinosa  Linn. Thwait (Araceae) is commonly known as “Spiny taro”, is a perennial herb that grows wild in marshy places throughout many parts of Asia. The Araceae family is used as traditional remedies or food and previous work on flavonoid content in the family showed that Araceae has a profile with flavone C-glycosides, flavonols, flavones, and proanthocyanidins as the main classes. Up to date, little information is available about the chemical constituents of  Lasia spinosa  Linn. Thwait (Araceae). Previously the inventors found that the hydro-alcohol extract exerted significant anti-cancer and anti-inflammatory activities. In this present invention, isolated major bioactivity compounds of  Lasia spinosa  (L.) Thwait and one proanthocyanidin were identified from the active extract. 
         [0004]    Citation or identification of any reference in this section or any other section of this application shall not be construed as an admission that such reference is available as prior art for the present application. 
       SUMMARY OF INVENTION 
       [0005]    Accordingly, the present invention relates to a therapeutic effect of proanthocyanidin A1 (PA1). More particularly, it relates to a compound that is naturally occurring in  Lasia spinosa  (L.) Thwait and its effects of treating an inflammatory disease or as an anti-cancer leading compound. 
         [0006]    In accordance with one aspect of the present invention, there is provided a method for treating an inflammatory disease by administering an effective dosage of a compound with the structure of: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    to a subject in need thereof. 
         [0007]    In a first embodiment of one aspect of the present invention, the compound is proanthocyanidin A1 (PA1). 
         [0008]    In a second embodiment of one aspect of the present invention, in the method for treating an inflammatory disease, the proanthocyanidin A1 (PA1) is isolated from a natural plant material comprising  Lasia spinosa  (L.) Thwait (Araceae). 
         [0009]    In a third embodiment of one aspect of the present invention, in the method for treating an inflammatory disease, the compound inhibits colitis, inhibits shortening of colon length, reduces colonic tissue damage, suppresses colonic myeloperoxidase activity (MPO) and/or inhibits nitric oxide (NO). 
         [0010]    In a fourth embodiment of one aspect of the present invention, in the method for treating an inflammatory disease, the effective dosage ranges from 0.81 mg/kg/day to 2.43 mg/kg/day. 
         [0011]    In a fifth embodiment of one aspect of the present invention, in the method for treating an inflammatory disease, the compound is administered orally. 
         [0012]    In a sixth embodiment of one aspect of the present invention, in the method for treating an inflammatory disease, the subject in need thereof is a human. 
         [0013]    Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. 
         [0014]    The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or more of the steps or features. 
         [0015]    Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. 
         [0016]    Furthermore, throughout the specification and claims, unless the context requires otherwise, the word “include” or variations such as “includes” or “including”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 
         [0017]    Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. 
         [0018]    Other aspects and advantages of the invention will be apparent to those skilled in the art from a review of the ensuing description. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0019]    The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which: 
           [0020]      FIG. 1  shows the structure of compound PAL 
           [0021]      FIG. 2  shows the wound healing assay of (a)-(d) control data; and (e)-(h) PA1 (cells were treated by compounds PA1 at 6.25 μg/mL which did not alter the cells viability); on human esophageal carcinoma (KYSE-150) cells. Original magnification, 5×. 
           [0022]      FIG. 3  shows the effects of PA1 on NO production in LPS-stimulated RAW 267.4 cells. After macrophages were treated with 100 ng/mL of LPS in the absence or presence of various concentrations of PA1 (1.56, 3.12, 6.25, 12.5, 25, 50 μg/mL), which did not alter the cells viability for 20 hours, the production of NO was determined by Griess reagent. Data were derived from three independent experiments and presented as mean±SEM ( #### p, compared with control group; *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001, compared with LPS-alone group). 
           [0023]      FIG. 4A  shows the effects of PA1 on the survival of mice. Colitis was induced in all groups except the control group. PA1 and SASP were administered to mice from day 6 to day 13. 
           [0024]      FIG. 4B ) shows change of body weight of mice. Colitis was induced in all groups except the control group. PA1 and SASP were administered to mice from day 6 to day 13. 
           [0025]      FIG. 4C  shows the disease activity index of mice. Colitis was induced in all groups except the control group. PA1 and SASP were administered to mice from day 6 to day 13. 
           [0026]      FIG. 4D  and  FIG. 4E  show the colon length of mice with DSS-induced colitis. Colitis was induced in all groups except the control group. PA1 and SASP were administered to mice from day 6 to day 13. The change in body weight was taken as the difference between the body weight before induction of colitis and that immediately before the mice were sacrificed on day 14. The DAI score was determined by combining the scores of (i) body weight loss, (ii) stool consistency, and (iii) stool bleeding. On day 14, the mice were sacrificed, and the colon lengths were measured. Data are expressed as mean±SEM, n=8 ( ## p, compared with control group; *p&lt;0.05 and **p&lt;0.01, compared with DSS group). 
           [0027]      FIG. 5  shows the effects of PA1 on histological manifestation in DSS-induced colitis in mice. (A) control group; (B) DSS model group; (C) DSS+SASP 200 mg/kg group; (D) DSS+PA1 10 mg/kg group; (E) DSS+PA1 30 mg/kg. Hematoxylin and eosin staining images of representative colons are shown at magnifications of 10×. 
           [0028]      FIG. 6  shows the effects of PA1 on suppressing myeloperoxidase (MPO) activity in the colon of mice with DSS-induced colitis. Colitis was induced in all groups except the control group. PA1 and SASP were administered to the mice from day 6 to day 13. On day 14, the mice were sacrificed, and MPO activity was determined from colon homogenates. Data are expressed as mean±SEM, n=8 (*p&lt;0.05, compared with DSS group). 
       
    
    
     DETAILED DESCRIPTION OF INVENTION 
       [0029]    The present invention is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only. 
         [0030]    The structures are identified by the  1 H, DEPT NMR spectroscopic data and the structure of proanthocyanidin A1 (PA1) is shown in  FIG. 1 . 
         [0031]    Proanthocyanidin A1: [M+H] + : 577.1364 (Calcd. for 577.1346).  1 H NMR (400 MHz, CD 3 OD): δ7.16 (1H, d, J=2 Hz, H-10), 7.04 (1H, dd, J=2, 8.5 Hz, H-14), 6.98 (1H, s, H-10′), 6.86 (1H, m, H-14′), 6.84 (1H, s, H-13), 6.82 (1H, s, H-13′), 6.10 (1H, s, H-6′), 6.08 (1H, d, J=2.4 Hz, H-8), 5.95 (1H, d, J=2.4 Hz, H-6), 4.76 (1H, d, J=8.0 Hz, H-8), 4.26 (1H, d, J=3.2 Hz, H-4), 4.15 (1H, d, J=3.2 Hz, H-3), 4.08 (1H, m, H-3′), 2.98 (1H, dd, J=5.6, 16.4 Hz, H-4′β), 2.59 (1H, dd, J=2, 8.5 Hz, H-4′α);  13 C NMR (100 MHz, CD 3 OD): 156.77 (s, C-7), 155.27 (s, C-5′), 154.77 (s, C-5), 152.70 (s, C-7′), 150.83 (s, C-8a), 149.45 (s, C-8′a), 145.42 (s, C-11), 145.37 (s, C-11′), 145.04 (s, C-12), 144.47 (s, C-12′), 130.83 (s, C-9), 129.55 (s, C-9′), 118.94 (d, C-14′), 118.46 (d, C-14), 114.96 (d, C-13), 114.34 (d, C-10), 114.25 (d, C-13′), 114.05 (d, C-10′), 105.16 (s, C-8′), 102.66 (s, C-4a), 101.44 (s, C-4a′), 99.03 (s, C-2), 96.77 (d, C-6), 95.21 (d, C-8), 95.16 (d, C-6′), 82.50 (d, C-2′), 67.00 (d, C-3′), 66.27 (d, C-3), 27.86 (d, C-4), 27.79 (t, C-4′). 
         [0032]    MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and DMSO (dimethyl sulfoxide), SASP (sulsafalazine), LPS (lipopolysaccharide, L3129), Griess reagent and all chemicals used were of HPLC grade from Sigma Chemical Co. (St. Louis, Mo., USA).  1 H NMR and  13 C NMR spectra were recorded on Bruker-Avance 400 MHz spectrometer, and CD 3 OD was used as solvent. Chemical shifts (δ) were reported in ppm with tetramethylsilane as an internal standard, and J values were given in Hz. High resolution mass spectra (HRMS) were performed on a VG Autospec-3000 spectrometer. Column chromatography was performed with HSCCC (high speed countercurrent chromatography), and the preparative HPLC were used. A waters 2535 Series machine equipped with Alltech Alltima-C 18  (4.6×250 mm, 5 μm) was used for HPLC analysis, and the preparative Alltech Alltima-C 18  (10×250 mm, 5 μm) was used in sample preparation. DSS (molecular weight: 36 to 50 kDa) was purchased from MP Biomedical (Santa Ana, Calif., USA). RPMI 1640 medium, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), FBS, penicillin and streptomycin were purchased from Life Technologies (Carlsbad, Calif., USA). 
         [0033]    Extraction and Isolation 
         [0034]    Air-dried pieces of  Lasia spinosa  (L.) Thwait root (500 g) were extracted three times with 75% EtOH (2 L) by reflex extraction at 60° C. The ethanol extract was concentrated to obtain a residue (23.1 g) which was then suspended in water (100 mL) in a separatory funnel prior to be partitioned by different solvents (3×100 mL). The n-butyl alcohol layer (3.5 g, 0.7%) was chromatographed on silica gel column (semi-preparative column Preparative RP-C 18 ) to obtain compound 1 (PA1, 6.16 mg). 
         [0035]    Cell culture 
         [0036]    Murine RAW264.7 macrophages and human esophageal cancer cell lines KYSE-70, KYSE-150, KYSE-410, KYSE-520 were maintained in RPMI 1640 or DMEM medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% FBS in an incubator at 37° C. with a humidified atmosphere of 5% CO 2 . Cells were sub-cultured every three days at a dilution of 1:6. 
         [0037]    Cytotoxicity Assay 
         [0038]    In the present invention, PA1 was dissolved in dimethyl sulfoxide (DMSO) to make stock solutions which were further diluted in culture medium for this experiment. Cells were seeded in a 96-well plate (3×10 3  cells/well) and allowed to attach to the plate overnight. After the recovery, cells were treated with 1.56, 3.125, 6.25, 12.5, 25, 50 μg/mL of PA1 in culture medium for 48 h. Then, 20 μL of MTT (5 mg/mL stock in PBS) per well was added into the medium (200 μL) and incubated for 4 h at 37° C. Finally, the culture medium was removed and 200 μL of DMSO were added to dissolve the purple formazan crystals. Absorbance of the solution was measured using a microplate reader spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, Calif.) at a wavelength of 570 nm. 
         [0039]    Wound Healing Assay 
         [0040]    6×10 4  cell/well were seeded in 12 well-plate at 30% confluence in completed medium. After 24 hours seeding, the monolayer was wounded by scoring with a sterile plastic tip (1 mL), then washed twice with PBS to remove cell debris and then incubated in a conditioned medium in the absence or presence of PA1 at 6.25 μg/mL for various periods of time up to 72 hours. Cell migration into the wound surface was monitored by Olympus IX71 microscopy and digitally photographed. 
         [0041]    Nitric Oxide Production Determination 
         [0042]    Nitric oxide (NO) production was indirectly assessed by measuring the nitrite levels in the cultured medium determined by a colorimetric method based on the Griess reagent. The cells were co-treated with various concentrations of PA1 in the absence or presence of LPS (100 ng/mL) at 37° C. for 24 hours. Then, 100 μL of each supernatant was mixed with the equal volume of Griess reagent incubated at room temperature for 15 minutes; meanwhile, sodium nitrite was used as a standard substance. The optical densities were measured at 540 nm with a micro-plate reader. 
         [0043]    Animals 
         [0044]    7-week-old male C57BL/6 mice weighing about 20-22 g were purchased from the Laboratory Animal Services Center, the Chinese University of Hong Kong. The study protocols were approved by the committee for Care of Laboratory Animals in the School of Chinese Medicine at the Hong Kong Baptist University. 
         [0045]    Induction of Chronic DSS Colitis and Treatment 
         [0046]    The animals were randomly assigned into five groups (n=8). Mice in the control group were supplied with distilled water whereas all other experimental groups were given 2.0% (w/v) dextran sulphate sodium (DSS) for 5 days. Thereafter, the mice of the DSS, SASP-treated (200 mg/kg/day) and PA1 (10 or 30 mg/kg/day)-treated groups were administered by gavage with saline, SASP or PA1 from day 6 to day 13, respectively. 
         [0047]    Human equivalent dosage is translated from mouse dosage using the following equation: D human =D mouse ×k (k=0.081) (Regan-Shaw et al. (2007)). Disclosure thereof is incorporated herein by its entirety.) Therefore, the human equivalent dosage is in a range of 0.81 mg/kg/day to 2.43 mg/kg/day. 
         [0048]    Evaluation of Disease Activity Index (DA1) 
         [0049]    The DAI was determined by scoring changes in the body weight, diarrhea, colon length, and bleeding. Each score was given in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Disease activity index scores based on disease marker intensities 
               
             
          
           
               
                   
                   
                 Body weight 
                   
                 Rectal 
               
               
                   
                 Score 
                 loss (%) 
                 Diarrhea 
                 bleeding 
               
               
                   
                   
               
               
                   
                 0 
                  &lt;1 
                 normal 
                 none 
               
               
                   
                 1 
                 1-5 
               
               
                   
                 2 
                  6-10 
                 Loose stools 
                 slight 
               
               
                   
                 3 
                 10-15 
               
               
                   
                 4 
                 &gt;15 
                 diarrhea 
                 fecal blood 
               
               
                   
                   
               
             
          
         
       
     
         [0050]    Histological Analysis 
         [0051]    The colons were dissected longitudinally, gently washed with ice-cold phosphate buffer saline (PBS), fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Each of five-micro-meter sections was stained with hematoxylin/eosin according to a standard procedure to evaluate colonic damage. The histological scoring system was shown in Table 2. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Histological scoring system for DSS-induced colitis 
               
               
                 scoring of severity of histological damage 
               
             
          
           
               
                 Score 
                 Description 
               
               
                   
               
               
                 0 
                 none 
               
               
                 1 
                 mild 
               
               
                 2 
                 moderate 
               
               
                 3 
                 severe 
               
               
                 0 
                 none 
               
               
                 1 
                 mucosa 
               
               
                 2 
                 mucosa and submucosa 
               
               
                 3 
                 transmural 
               
               
                 0 
                 none 
               
               
                 1 
                 ⅓ damaged 
               
               
                 2 
                 ⅔ damaged 
               
               
                 3 
                 crypt loss by surface epithelium present 
               
               
                 4 
                 Both crypt and surface epithelium lost 
               
               
                   
               
             
          
         
       
     
         [0052]    Determination of Neutrophil Infiltration in Colon Tissue (MPO Assay) 
         [0053]    Myeloperoxidase (MPO) is an enzyme mainly released by neutrophil, and its activity is directly associated with the severity of inflammation in a given tissue. In the present invention, MPO activity was measured as described in the inventor&#39;s earlier publication (Mu, H. X., et al.,  Anti - inflammatory Actions of  (|)-3′ alpha - Angeloxy -4′- keto -3′,4′- dihydroseselin  ( Pd - Ib )  against Dextran Sulfate Sodium - Induced Colitis in C 57 BL/ 6  Mice . J Nat Prod, 2016). The results were normalized to the wet weight of colon tissue and quantified as units/g tissue. 
         [0054]    Discussion 
         [0055]    In the present invention, the n-butyl alcohol extract of root of  Lasia spinosa  (L.) Thwait showed potential anti-cancer and anti-inflammatory activities. The inventors purified the active fraction using a column chromatography of the preparative HPLC to obtain a proanthocyanidin. 
         [0056]    To determine the cytotoxic effect of PA1 against human esophagus cancer cells (KYSE-70, KYSE-150, KYSE-450 and KYSE-520 cell lines), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was performed. 
         [0057]    Furthermore, proanthocyanidin A1 (PA1), was found to be more cytotoxic against KYSE-450 cells than other cell lines (Table 3). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Cytotoxicity of compounds against cancer cell lines 
               
             
          
           
               
                 Compound 
                 KYSE-150 
                 KYSE-70 
                 KYSE-520 
                 KYSE-450 
               
               
                   
               
               
                 PA1 
                 — 
                 — 
                 — 
                 48.64 
               
               
                   
               
               
                 Results are expressed as IC 50  values in μg/mL 
               
             
          
         
       
     
         [0058]    In addition, wound-healing assay was used to test whether those compounds could affect cell motility. A confluent monolayer of KYSE-150 cells was scratched to form a wound and incubated in the absence or presence of PA1 at 6.25 μg/mL. The results obtained were compared to that of controls. After 72 hours, the wound edges were indistinguishable in control group, while compound-treated cells did not migrate into the wound ( FIG. 2 ), indicating that PA1 had an anti-migration effect on KYSE-150 cells in the absence of cytotoxicity. 
         [0059]    The effect of PA1 on nitric oxide (NO) production was investigated using Griess reagent. As shown in  FIG. 3 , stimulation with LPS (100 ng/mL) resulted in a significant increase in NO production compared with the control group while treatments with PA1 at different concentration led to significant inhibition of NO production. 
         [0060]    To determine the role of PA1 in inflammatory bowel disease (IBD), a DSS-induced colitis mouse model was used. In the present invention, PA1 treated mice showed reduced body weight loss and had a more rapid recovery that those mice exposed to 2% DSS in drinking water for 5 days ( FIG. 4B ). DAI scores indicated that PA1 also led to a clinical improvement in DSS-induced colitis ( FIG. 4C ). In addition, as another important symptomatic parameter in DSS-induced colitis, shortening of colon length, was rectified after administration with PA1 ( FIG. 4D and 4E ). Mortality decreased in the PA1 treated group, and the results are consistent with the reduction in mucosal ulceration, crypt damage, edema, and cell infiltration into mucosal tissue when compared with the DSS group ( FIG. 4A  and  FIG. 5 ). Enzyme myeloperoxidase (MPO) reflects the neutrophil infiltration in colon tissue damage. PA1 treatment was found to significantly lower the MPO activity ( FIG. 6 ). 
         [0061]    In this invention, PA1 may ameliorate inflammation in colitis and exert significant anti-tumor invasion activity in esophageal cancer cell line. 
         [0062]    In summary, PA1 can be developed as a novel therapeutic agent of colitis and cancer. 
       INDUSTRIAL APPLICABILITY 
       [0063]    The present invention discloses a chemical entity isolated from natural sources for its therapeutic and medical uses. More particularly, it relates to compound that is naturally occurring in the plant of  Lasia spinosa  Linn. Thwait (Araceae) and its biological activity of treating an inflammatory disease or as an anti-cancer leading compound. 
         [0064]    If desired, the different functions discussed herein may be performed in a different order and/or concurrently with each other. Furthermore, if desired, one or more of the above-described functions may be optional or may be combined. 
         [0065]    While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the reminder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extend. All publications recited herein are hereby incorporated by reference in their entirety.