Patent Publication Number: US-2006013900-A1

Title: Use of betel leaf extract to induce IFN-gamma production from human peripheral blood T cells and as a Th1 type immunomodulator

Description:
FIELD OF THE INVENTION  
      This invention relates to use of betel leaf extract to induce IFN-gamma production from human peripheral blood T cells.  
     BACKGROUND AND PRIOR ART REFERENCES  
      Stinging nettle leaf extracts are registered in Germany for adjuvant therapy of rheumatic diseases. In a whole blood culture system, the nettle extract IDS 23 (Rheuma-Hek) inhibited lipopolysaccharide stimulated monocyte cytokine expression, indicating an immunomodulating effect. (Antirheumatic Effect of IDS 23, a Stinging Nettle Leaf Extract, on in vitro expression of T helper cytokines). The applicants investigated the immunomodulating effects of betel leaf extracts on phytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) in vitro. Betel leaves has a strong pungent aromatic flavor and widely used as a masticatory. Generally, mature or over mature leaves, which has ceased growing but not yet become brittle are used for chewing. The basic preparation for chewing purposes consists of betel leaf smeared with hydrated lime and catechu to which scrapings of arecanut are added; flavorings such as coconut shavings, clove, cardamon, fennel, powdered liquorice, nutmeg and also tobacco are used according to one&#39;s taste. In some places prepared pan is covered with silver or gold leaf. As a masticatory, it is credited with many properties: it is aromatic, digestive, stimulant and carminative. Medicinally it is useful in catarrhal and pulmonary affections; it is also used for poultices. The effects of chewing of betel with arecanut and other adjuncts are the excitation of the salivary glands and the irritation of the mucous membrane of the mouth. The red coloration produced is due to a pigment in the arecanut, which manifests itself under the action of alkali in lime and catechu. A mild degree of stimulation is produced, resulting in a sensation of warmth and well being, besides imparting a pleasant odor. The most important factor determining the aromatic value of the leaf is the amount and particularly the nature of the essential oil present. Betel leaves from different regions vary in smell and taste. The most pungent is the  Sanchi  type, while the most mild and sweet ones are from Madras. The betel leaves contain essential oils, the content of oil varies from 0.7 to 2.6 per cent depending upon the varieties of leaves. The oil consists of phenols and terpens. The higher the proportion of phenol oil, the better the quality. An isomer of eugenol named chavibetol (betel phenol; 4-allyl-2-hydroxy-1-methoxy benzene) is considered to be the characteristic constituent of betel oil. It is however, absent in Indian samples. Betel oil of Indian types contain as a predominant phenolic constituent. Oil of betel has been used in the treatment of various respiratory catarrhs, under as a local application either by gargle or by inhalation in diphtheria. It has carminative properties. It exhibits in different action on the central nervous system of mammals; lethal doses produce deep narcosis leading to death within a few hours. The essential oil and extracts of the leaves possess activity against several Gram-positive and Gram-negative bacteria such as  Micrococcus pyogenes  var.  albus  and var.  aureus, Bacillus subtilis  and  B. megaterium, Diplococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Salmonella typhosa, Vibrio comma, Shigella dysenteriae, Proteus vulgaris, Pseudomonas solanacaerum, Sarcina lutea  and  Erwinia carotovora.  The oil is found to be lethal in about 5 minutes to the protozoa  Paramaecium caudatum  (Wealth of India, Vol.8 pp. 84-94). It inhibits the growth of  Vibrio cholerae, Salmonella typhosum  and  Shigella flexneri  and  Escherichia coli.  Steam—distillate of the leaves showed activity against  Mycobacterium tuberculosis.    
     OBJECTS OF THE INVENTION  
      The main object of the present invention is to provide a method for inducing IFN-γ from human peripheral blood mononuclear cells.  
      Another object of the present invention relates to use of betel leaf extract for inducing IFN-γ production from human peripheral blood T cells.  
      Another object of the invention is to provide a composition comprising betel leaf extract, which is useful as Th 1  type immunomodulator.  
     SUMMARY OF THE INVENTION  
      To meet the above objects, the present invention provides a method for inducing IFN-γ from human peripheral blood mononuclear cells. The invention also relates to use of betel leaf extract for inducing IFN-γ production from human peripheral blood T cells.  
     DETAILED DESCRIPTION OF THE INVENTION  
      Accordingly, the invention relates to a method for inducing IFNγ from human peripheral blood mononuclear cells wherein the said method comprising preparing water extract of betel leaf, preparation of human peripheral blood mononuclear cells; incubation of hPBMC with betel leaf extract for a period of 18-48 hours; extraction of RNA for cytokine specific RT-PCR or for flow cytometry for the detection of intracellular cytokine protein.; subjecting RNA for RT-PCR to obtain PCR products using IFNγ specific known primers and enhancement of IFNγ as reflected by IFNγ specific band.  
      Alternatively, one more method for inducing IFNγ produced from human peripheral blood mononuclear cells wherein the said process comprising subjecting incubated cells for intracellular staining for IFNγ; analysis of stained cells in flow cytometer; and enhancement of IFNγ positive cells to at least seven fold.  
      A method for using betel leaf extract as Th 1  type immuno-modulator wherein the said method comprising: 
          a) administering to a subject at least 5 to 10 mg/ml/kg body wt. of betel leaf extract, and     b) administering the extract through oral or intramuscular route once in a day for a period of at least one month,        

      In an embodiment of the present invention provides a pharmaceutical composition useful as Th 1  type immunomodulator, said composition comprising an effective amount of betel leaf aqueous extract or lyophilized betel leaf extract together with or associated with an additive.  
      In still another embodiment of the invention, the additive is selected in such a manner that does not interfere with the activity of betel leaf extract.  
      Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.  
      Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.  
      Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.  
      Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10  
      Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.  
      In one more embodiment of the present invention, a method of treating a subject to provide Th1 type immuno-mudulation, said method comprising administering a pharmaceutically effective amount of betel leaf extract, lyophilized extract or a composition comprising the extract to the subject.  
      Yet another embodiment of the present invention, the additive is selected in such a manner it does not interfere with the activity of lyophilized betel leaf extract.  
      Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.  
      Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.  
      Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.  
      Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10  
      Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.  
      In one more embodiment of the present invention, use of betel leaf extract in association with suitable carriers/additive as Th 1  type immunomodulator.  
      Yet another embodiment of the present invention, the additive is selected in such a manner it does not interfere with the activity of lyophilized betel leaf extract. Yet another embodiment of the invention, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.  
      Yet another embodiment of the invention, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.  
      Yet another embodiment of the invention, the oral route is in the form of capsule, syrup, concentrate, powder or granules.  
      Yet another embodiment of the invention, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10  
      Yet another embodiment of the invention, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month. One more embodiment of the present invention relates to the preparation of betel leaf extracts comprising the following steps; 
          1) washing of the fresh leaves of  Piper betel  and homogenizing in a mixture blender;     2) sonicating in an ultrasonic bath with 2 to 3 bursts each for 15 minutes and filtering the extract, if desired repeating the extraction at least once and drying; and     3) lyophilizing the extract to get a semi-solid mass        

      In one more embodiment of the invention, the lyophilized extract is obtained by freeze drying the aqueous extract by conventional methods.  
      In another embodiment, the water extract is prepared from following types of betel leaf ( Piper betel ) namely Wild type, Climber type, Bangla type and Sweet type. 
    
    
     BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS  
       FIG. 1 : represents RT-PCR to demonstrate that betel leaf extract enhances IFN-γ mRNA expression by peripheral blood mononuclear cells (PBMC) of normal individuals.  
       FIG. 2 : represents flow cytometric determination that betel leaf extract enhances IFN-γ expression at the protein level by PBMC of normal individuals. 
    
    
      The following examples further illustrate the invention, but the invention is not limited thereto.  
     EXAMPLE 1  
      34.14 gm of fresh leaves of  Piper betle  thoroughly washed in sterile water was homogenized with 100 ml of glass distilled water in a mixture-blender. It was then sonicated in an ultrasonic bath with 3 burst each for 15 min. The extract was filtered through Whatman No.1 filter paper and the filtrate was collected. This process of extraction was repeated three times. The combined extract was lyophilized yielding a semi-solid mass weighing 1.17 gm. This was then tested for biological activity.  
     EXAMPLE 2  
      The fresh leaves of  Piper betle  weighing 21.68 gm homogenized with distilled water (60 ml) in a mixture—blender and then sonicated in an ultrasonic bath with 2 burst each for 15 min. It was allowed to be extracted overnight or 16 hours. Filtering through Whatman No. 1 filter paper separated the material extracted in water. This type of treatment for extraction was repeated for three times. The combined extract was evaporated to dryness in a flash evaporator under reduced pressure at 45° C. The residual substance was then dried in a desiccator under high vacuum and the semi-solid mass weighing 0.59 gm was tested for biological activity.  
      Properties of the Materials  
      The biologically active material obtained by examples 1 and 2 has the following properties: 
          1) The dried semisolid prepared as stated above was a dark colored material soluble in water and dimethyl sulfoxide.     2) Thin layer chromatography of the active material shows five spots having R f  0.75, 0.64, 0.50, 0.40 and 0.33 in the solvent system of n-butanol, acetic acid and water in the ratio of 9:5:7 respectively.     3) The HPLC analysis of the active material using Intersil ODS-3 (4.6×250 mm) analytical column, solvent system methanol and water in the ratio of 4:1 and a flow rate of 1.0 ml/min., detection at 217 nm resolved the material into eleven peaks with the retention time of 2.69, 4.27, 5.95, 6.97, 7.49, 9.39, 11.20, 12.40, 15.53, 18.90 and 21.49 mins.        

     EXAMPLE 3  
       1 . Preparation of Human Peripheral Blood Mononuclear Cells (PBMC):  
      Heparinized whole bloods (collected from normal individuals) were subjected to Ficoll Hypaque density gradient centrifugation. Cells in the interface were washed twice with phosphate buffered saline (PBS) and then re-suspended in medium RPMI-1640 supplemented with 10% Fetal Bovine Serum.  
      2. Incubation of hPBMC with Betel Leaf Extract:  
      PBMC (5.0×10 6  cells) were cultured overnight (18 hours) at 37° C. in 5% CO 2  in a total volume of 2.0 ml RPMI+10% FBS containing 5 μg/ml of phytohemagglutinin (PHA) in 24 well plates in the presence or absence of betel leaf extracts (12.5 mg/ml final concentration). At the end of the incubation period PBMC were washed twice with PBS and used for extraction of total RNA for cytokine specific RT-PCR or for flow cytometry for the detection of intracellular cytokine at the single cell level.  
      3. RNA Preparation and RT-PCR  
      Total cellular RNA from cultured PBMC was extracted by Trizol (Gibco BRL). 5×10 6  cells were cultured for 18 hours as described above and harvested and resuspended in 1 ml Trizol. 2 μg of RNA and 10 μm of each primers were used to synthesize cDNA and PCR in single tube using superscript TM One step RT-PCR system (Gibco BRL) in a total volume of 25μl-Primer sequences of human IFNγ and human IL-4 were as follows:  
      IFNγ sense, 5′ TCT GCA TCG TTT TGG GTT CT 3′, antisense 5′ CAG CTT TTC GAAGTC ATC TC 3′; IL-4 Sense 5′ CCT CTG TTC TTC CTG CTAGC 3′; antisense 5′ CCG TTTCAGGAA TCG GAT CA 3′. Amplification and cDNA synthesis was performed as described in manufacturer&#39;s protocol. PCR products were electrophoresed in a 1.2% agarose gel in the presence of ethidium bromide and photographed under ultraviolet transmilluminator. Molecular weight markers (123 bp ladder, Gibco BRL) were included in all gels. (J. Rheumatol. 1999: 26: 2517-2522)  
      4. Flow Cytometry  
      Cells were washed, permeabilized by treatment with 4% paraformaldehyde for 10 min., followed by incubation with 0.1% saponin for 10 min. Cells were then washed with washing buffer (PBS containing 1% albumin, 0.1% saponin and 0.1% sodium azide). After washing, permeabilized cells were treated with FITC or PE labeled control monoclonal antibody (mAb), anti-human IFNγ Ab labeled with FITC or anti-human IL-4 mAb labeled with PE, for 20 min. in room temperature at dark. Cells were then washed once with washing buffer and once with PBS and then resuspended in PBS containing 1% paraformaldehyde for flow cytometric analysis.  
      Results:  
      Influence of Betel Leaf Extract on Th 1  Cytokine Expression  
      It is clear that  FIG. 1 , that betel leaf extract enhances IFNγ mRNA expression but has no effect on IL-4 m RNA expression by peripharal blood mononuclear cells of normaL human individuals as determined by RT-PCR. In other words, the applicants&#39; data as shown in Fig. I indicates that betel leaf extract significantly enhances synthesis of IFNγ specific mRNA and having virtually no effect on IL-4 mRNA sysnthesis.  
      IFNγ synthesis by PBMC was also detectable at the protein level as evident from the flow cytometric dot plot analysis as shown in Fig. II. Only 0.9% PBMC were positive for IFNγ when PBMC were incubated with PHA (Fig. IIA). On the other hand, 7.1% PBMC showed intracellular IFNγ (Fig II B) when PBMC were incubated with PHA plus betel leaf extract. In contrast, percentages of IL-4 producing cells were not appreciably changed when PBMC were incubated with betel leaf extract (Fig. II C &amp; D).  
      Discussion  
      T cells are subdivided into Th 1  and Th 2  phenotypes by their cytokine patterns (Mossmann, T. R., Coffman, R L. Th 1  and Th 2  cells: different patterns of lymphokine secretions lead different functional properties. Annual Rev. Immunol. 1989, 7; 145 -73), which regulate cell mediated and humoral immune responses. Inflammatory immune responses are primarily mediated by Th 1  cell polpulations through the production of IL 2  &amp; 1FN-γ, which enhance cellular immunity (Trinchieri G. Interlenkin—12 and its role in generation of Th 1  cells. Immunol Today 1993; 14: 335-8; Germann, T., Szeliga, J., Hess, H. et al. Administration of interlenkin—12 in combination with type-II collagen induces severe arthritis in DBA/1 mice. Proc Natl Acad Sci. USA 1995; 92: 4823-7).  
      Thus, our experimental results suggest that betel leaf extracts enhance Th 1  type response leading to enhanced cellular immunity.