Extract of Asplenium nidus L

Abstract

An extract of Asplenium nidus L. includes pyropheophorbide a methyl ester (C.sub.34H.sub.36N.sub.4O.sub.3), pheophorbide a methyl ester (C.sub.35H.sub.36N.sub.4O.sub.5), 1-linleoyl-3-linolenoyl-glycerol (C.sub.39H.sub.66O.sub.5), 1-linleoyl-2,3-dipalmitoyl-rac-glycerol (C.sub.53H.sub.98O.sub.6) and 1,3-dipalmitoyl-sn-glycerol (C.sub.35H.sub.68O.sub.5). The extract of Asplenium nidus L. is obtained by using a method including steps of: solvent extraction, using an solvent to extract an Asplenium nidus L. sample and to obtain an extract, with a w/v ratio between the solvent and the Asplenium nidus L. sample being 50˜60 mg/ml; and column chromatography, fractionating the extract with water and ethanol as eluent to obtain several fractions including fraction a to fraction i, and further evaporating and lyophilizing fraction b, c, d or their combination to obtain an extract of Asplenium nidus L.

Claims

1. A method of treating benign prostate hyperplasia in a subject in need thereof comprising: administering an effective amount of an extract of Asplenium nidus L. to the subject, wherein the extract comprises: pyropheophorbide a methyl ester; pheophorbide a methyl ester; 1-linleoyl-3-linolenoyl-glycerol; 1-linleoyl-2,3-dipalmitoyl-rac-glycerol; and 1,3-dipalmitoyl-sn-glycerol.

2. The method as claimed in claim 1, wherein the extract of Asplenium nidus L. is in the form of a health product.

3. The method as claimed in claim 1, wherein the effective amount of the extract of Asplenium nidus L. is 10-50 mg/kg of body weight of the subject in need thereof.

4. The method as claimed in claim 1, wherein the effective amount of the extract of Asplenium nidus L. is 50 mg/kg of body weight of the subject in need thereof.

5. A method of treating prostatitis in a subject in need thereof comprising: administering an effective amount of an extract of Asplenium nidus L. to the subject, wherein the extract comprises: pyropheophorbide a methyl ester; pheophorbide a methyl ester; 1-linleoyl-3-linolenoyl-glycerol; 1-linleoyl-2,3-dipalmitoyl-rac-glycerol; and 1,3-dipalmitoyl-sn-glycerol.

6. The method as claimed in claim 5, wherein the extract of Asplenium nidus . is in the form of a health product.

7. The method as claimed in claim 5, wherein the effective amount of the extract of Asplenium nidus L. is 10-50 mg/kg of body weight of the subject in need thereof.

8. The method as claimed in claim 5, wherein the effective amount of the extract of Asplenium nidus L. is 50 mg/kg of body weight of the subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) The present invention will become more fully understood from the detailed description given hereinafter and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:

(3) FIG. 1 is a diagram illustrating the chemical structure of pyropheophorbide a methyl ester.

(4) FIG. 2 is a diagram illustrating the chemical structure of pheophorbide a methyl ester.

(5) FIG. 3 is a diagram illustrating the chemical structure of 1-linleoyl-3-linolenoyl-glycerol.

(6) FIG. 4 is a diagram illustrating the chemical structure of 1-linleoyl-2,3-dipalmitoyl-rac-glycerol.

(7) FIG. 5 is a diagram illustrating the chemical structure of 1,3-dipalmitoyl-sn-glycerol.

(8) FIG. 6 is a diagram illustrating an extracting method of an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(9) FIG. 7 is a diagram illustrating eluting fractions of an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(10) FIG. 8 is diagram illustrating another extracting method of an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(11) FIG. 9 is a bar chart illustrating the level of PSA-Luc inhibition of an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(12) FIG. 10 is another bar chart illustrating the level of PSA-Luc inhibition of an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(13) FIG. 11 is a bar chart illustrating cell viability of WPMY-1 after treated with an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(14) FIG. 12 is a bar chart illustrating cell viability of LNCaP after treated with an extract of Asplenium nidus L. in a preferable embodiment of the present invention.

(15) FIG. 13 is a bar chart illustrating cell viabilities of WPMY-1 and LNCaP under various dosage of an extract of Asplenium nidus L. in the present invention.

(16) FIG. 14a is a chart illustrating prostatic index in BPH mice under various dosage of an extract of Asplenium nidus L. in the present invention.

(17) FIG. 14b is a chart illustrating excretion of urine in BPH mice under various dosage of an extract of Asplenium nidus L. in the present invention.

(18) FIG. 14c is a chart illustrating water intake in BPH mice under various dosage of an extract of Asplenium nidus L. in the present invention.

(19) FIG. 15 is morphological aspects (to vi, H & E staining; Masson's trichrome staining, vii to ix, ×100) of the disease-free (i) & (vii), phenylephrine-stimulated (ii) and (viii), and samples-treated (iii: ANE; iv and v: Lo; vi and ix: Hi) ventral prostate in BPH mice under various dosage of an extract of Asplenium nidus L. in the present invention.

(20) In the various figures of the drawings, the same numerals designate the same or similar parts. Furthermore, when the terms “first”, “second”, “third”, “fourth”, “inner”, “outer” “top”, “bottom” and similar terms are used hereinafter, it should be understood that these terms have reference only to the structure shown in the drawings as it would appear to a person viewing the drawings, and are utilized only to facilitate describing the invention.

DETAILED DESCRIPTION OF THE INVENTION

(21) With reference to FIGS. 1 and 2, a preferred embodiment of the invention discloses an extract of Asplenium nidus L. comprising pyropheophorbide a methyl ester (C.sub.34H.sub.36N.sub.4O.sub.3; as shown in FIG. 1), pheophorbide a methyl ester (C.sub.35H.sub.36N.sub.4O.sub.5; as shown in FIG. 2), 1-linleoyl-3-linolenoyl-glycerol (C.sub.39H.sub.66O.sub.5, as shown in FIG. 3), 1-linleoyl-2,3-dipalmitoyl-rac-glycerol (C.sub.53H.sub.98O.sub.6, as shown in FIG. 4) and 1,3-dipalmitoyl-sn-glycerol (C.sub.35H.sub.68O.sub.5, as shown in FIG. 5) and the said extract of Asplenium nidus L. is capable of inhibiting the secretion of androgen, decreasing inflammatory and reducing incidence to prostate disease.

(22) FIG. 6 shows an extracting method of the extract of Asplenium nidus L. of the preferable embodiment in the present invention, comprising steps of solvent extraction S1 and column chromatography S2.

(23) In the step of solvent extraction S1, a sample of Asplenium nidus L. is prepared and extracted with a solvent, with a w/v ratio of the sample of Asplenium nidus L. and the solvent being 50-60 mg/ml, to obtain an extract, wherein the solvent is 95% alcohol.

(24) In the step of column chromatography, the said extract is fractioned by using a silica gel column chromatography, with water and alcohol being eluents, to obtain several fractions, including fractions a to i by order of elution, and then fractions b, c, d or their combination is selected, evaporated, and lyophilized to obtain the extract of Asplenium nidus L. of the preferable embodiment of the present invention.

(25) Precisely, elution in the step of column chromatography is carried out via an automated middle-sized liquid chromatography-Flash LC, preferably with reverse phase column C18, to achieve preferable extracting rate, followed by using eluents in various compositions to obtain several fractions, as shown in FIG. 7. The several fractions comprising fractions a, b, c, d, e, f, g, h, i by the order of elution are successfully obtained. Finally, the fractions b, c, d or their combination are selected, evaporated and lyophilized to obtain the extract of Asplenium nidus L. of the preferable embodiment in the present invention. The said eluents comprise alcohol and water mixed with each other in a linear gradient, wherein a concentration of alcohol in the eluents varies from 70% to 100%.

(26) With reference to FIG. 8, the extracting method of the extract of Asplenium nidus L. of the preferable embodiment in the present invention preferably comprises a step of selection S3 before the step of solvent extraction S1, with the sample of Asplenium nidus L. being selected as a whole plant of Asplenium nidus L. (including roots), so that, an extract of Asplenium nidus L. having a great amount of active substance is successfully obtained. Furthermore, the sample of Asplenium nidus L. is selected from a whole plant of Asplenium antiquum Makino or Asplenium nidus Linn, preferably a whole plant of Asplenium nidus Linn including roots. Since, Asplenium nidus Linn is commonly distributed and cultivated throughout Taiwan, throughout the year, it is easy to obtain the sample of Asplenium nidus L. and further to use in the extracting method of the present invention. Also, the Asplenium nidus Linn is green and organic vegetables, being free from contamination of pesticide and insects, so that the food safety thereof can be guarantee.

(27) It is demonstrated that the extract of Asplenium nidus L. has active substances (including pyropheophorbide a methyl ester, pheophorbide a methyl ester, 1-linleoyl-3-linolenoyl-glycerol, 1-linleoyl-2,3-dipalmitoyl-rac-glycerol and 1,3-dipalmitoyl-sn-glycerol) against androgen and inflammation. Therefore, the said extract of Asplenium nidus L. is capable of modulating the secretion of hormone, and further controlling diseases caused by hormone imbalance, such as prostate diseases (including benign prostate hyperplasia, prostate cancer and prostatitis), incontinence, androgenic alopecia and menopausal disorders.

(28) Also, the extract of Asplenium nidus L. is easy to obtain and to isolate, capable of being used on pharmaceutical industry, as an active substance in medications or health products for the said disease, the said prostate diseases in particular. The extract of Asplenium nidus L. can be used individually, or in combination with pharmaceutical acceptable vehicles, excipients, salts or other nutrients, being in a composite. In addition, the extract of Asplenium nidus L. can be further manufactured into any oral type that is easy to take, such as pastil, capsule, powder, pill, solution, or fermented products. Yet, the extract of Asplenium nidus L. can be combined with other food products or drinks, being manufactured into a more convenient type for taking.

(29) For proving the extract of Asplenium nidus L. in the preferable embodiment of the present invention truly having ability against androgen, a serial of trial is demonstrated below. However, the application of the said extract of Asplenium nidus L. is not limited to that.

(30) In the present trial, the said extracting method is carried out by processing the step of selection S3, the step of solvent extraction S1, and the step of column chromatography S2 sequentially, with the sample of Asplenium nidus L. being extracted by using 95% alcohol (being the solvent), followed by eluting via the automated middle-sized liquid chromatography-Flash LC (with reverse phase column C18) to obtain 9 fractions (including a to i). With reference to FIGS. 9 and 10, the 11 fractions are collected and cultured with prostate cancer cell line 22Rv/103E respectively, or co-cultured with a mix of the prostate cancer cell line 22Rv/103E and stromal myofibroblast-WPMY-1 respectively, and then activity against androgen of each fraction is determined.

(31) In view of FIGS. 9 and 10, fractions b, c, and d all show significant inhibition either when they are cultured with the prostate cancer cell line 22Rv/103E respectively (see FIG. 9), or are co-cultured with the mix of the prostate cancer cell 22Rv/103E and the stromal myofibroblast-WPMY-1 (see FIG. 10), and particularly in a dose-dependent manner.

(32) In FIGS. 11 and 12, cell viability of the stromal myofibroblast-WPMY-1 and prostate cancer cell line-LNCaP are demonstrated after culturing with the 9 fractions respectively. The stromal myofibroblast-WPMY-1 and the prostate cancer cell line-LNCaP are cultured with the 9 fractions respectively for 48 hours. After that, the stromal myofibroblast-WPMY-1 and the prostate cancer cell line-LNCaP are dyed with SRB dye, and then the cell viability thereof is determined.

(33) According to FIGS. 11 and 12, the fractions b, c, and d apparently have inhibition on the growth of the stromal myofibroblast-WPMY-1 and the prostate cancer cell line-LNCaP in a dose-depended manner.

(34) With reference to FIG. 13, the stromal myofibroblast-WPMY-1 (1) and the prostate cancer cell line-LNCaP (2) are cultured with a composite of the fractions b, c, and d, at various dosages respectively. After that, cell viabilities of the stromal myofibroblast-WPMY-1 and the prostate cancer cell line-LNCaP are determined after the culturation, by using colony formation assay. In FIG. 13, it is noted that the composite has inhibition against the growth of the stromal myofibroblast-WPMY-1 (see line 1) and the prostate cancer cell line-LNCaP (see line 2) in a dose-depended manner. Also, a higher dosage of the composite, such 100 μg/mL, will lead to the death of prostate cancer cells. In the present trial, the IC.sub.50 of the composite on the stromal myofibroblast-WPMY-1 and the prostate cancer cell line-LNCaP 11.63 μg/mL and 13.19 μg/mL respectively.

(35) Next, for proving the extract of Asplenium nidus L. in the preferable embodiment of the present invention truly having ability improving against androgen, a serial of trial is demonstrated below. However, the application of the said extract of Asplenium nidus L. is not limited to that.

(36) C57BL/6Jnarl male mice purchased from the animal center of the National Laboratory Animal Center (NLAC, Taiwan) are used in this experiment. The mice are housed in a SPF animal room of Agricultural Biotechnology Research Center, Academia Sinica, Taiwan with constant temperature of 22° C. where is kept on a 12-hours light and 12-hours dark cycle.

(37) BPH mice are induced by chronic α(1)-adrenergic stimulation (15 mg/kg of body weight of the mice) and daily subcutaneous (sc) injection of phenylephrine (PE) 5 days/week for five weeks. Furthermore, the BPH mice are treated with different doses of the extract of Asplenium nidus L. of the invention. Prostate index (PI, ventral prostate wet weight/mouse weight), urine excretion and water intake of the BPH mice are recorded in FIGS. 14a, 14b and 14c. Moreover, the BPH mice are sacrificed and morphological aspects of ventral prostate are shown in FIG. 15.

(38) As shown in FIG. 14a, compared with group control, group PE (treated with phenylephrine) has a significant increased PI value indicating the enlargement of prostate. The group PE excretes a significantly lower level of urine and an amount of water intake of group PE is significantly decreased over the group control. On the other hand, in group ANE (treated with ethanolic extract of Asplenium nidus L. without step of column chromatography, 100 mg/kg of body weight/day), group Hi (treated with the extract of Asplenium nidus L. of the invention, 50 mg/kg of body weight/day) or group Lo (treated with the extract of Asplenium nidus L. of the invention, 10 mg/kg of body weight/day), the PI, urine excretion and water intake return to the normal level (ρ<0.01, vs. PE group). The group Hi has a preferable effect if the PI, urine excretion and water intake (as shown in FIGS. 14a, 14b and 14c). As a result, the extract of Asplenium nidus . of the invention is capable of improving BPH phenomenon.

(39) The groups control, PE, ANE, Lo and Hi are sacrificed and Hematoxylin-eosin staining (H&E staining) and Masson's trichrome staining of ventral prostate are preformed. In the H&E staining, compared with the group control, symptom of epithelial hyperplasia occurred in the group PE and the luminal areas of the ducts are decreased, suggesting a compression of secretory gland (as shown in FIG. 15-ii). The groups ANE, Lo and Hi are improved in the symptom of epithelial hyperplasia (as shown in FIG. 15-iii, iv and vi).

(40) As shown in FIG. 15-vii-xi, collagen distributions of each group are shown in the Masson's trichrome staining. The group control (FIG. 15-vii) shows higher area of collagen (blue area, yellow arrows) and reticular fibers in the stromal region (orange arrows) than the group PE (FIG. 15-viii). Treated with the extract of Asplenium nidus L. of the invention (the group Hi) presents the restoration of collagen area and reticular fibers (FIG. 15-ix).

(41) Therefore, the extract of Asplenium nidus L. of the invention is capable of improving PE-indued BPH phenomenon, increasing urine excretion and further improving the pathological characteristics of BPH. The extract of Asplenium nidus L. of the invention can be used as an active substance in a form of drug or healthy product treating of prostate disease.

(42) Further, a practical example of the said extract of Asplenium nidus L. is provided below. However, the practical use of the said extract of Asplenium nidus . is not limited to that.

(43) In the present trial, the said extracting method is carried out by processing the step of selection S3, the step of solvent extraction S1, and the step of column chromatography S2 sequentially, to extract the sample of Asplenium nidus L. and to obtain the extract of Asplenium nidus L. With reference to Table 1, the obtained extract of Asplenium nidus L. is given to 88 patients (in various age groups) that suffer from various prostate diseases (including benign prostate hyperplasia, prostate cancer or prostatitis). Obviously, the extract of Asplenium nidus L. is sufficient to be used on the therapy of benign prostate hyperplasia, prostate cancer and prostatitis, with symptoms of urination being improved in 2-4 weeks in average and without leading to any side effects.

(44) Generally, one of primary symptoms of benign prostate hyperplasia is urorrhagia and nocturn. However, with treatment of the said extract of Asplenium nidus L., frequencies of urophagia and nocturnal enuresis on patients are dramatically reduced till the same as normal. Moreover, according to International Prostate Symptom Score (I-PSS), the said 88 patients has around 20-30 points of improvement in 2-4 weeks. In comparison with other commercial health products (comprising 6% lycopene), only 2-3 points of improvement can be achieved.

(45) TABLE-US-00001 TABLE 1 Therapeutic Effects on 88 Patients Ages Effects Numbers .sup. 93~101 Normal urination, do not need 1 further treatment 70~85  Normal urination, do not need 8 further treatment 45~80  Normal urination 69 Normal urination, levels of PSA 2 reduce from 8~9.5 to 6.0 in a short time 55 Normal urination in three days 1 55~60.sup.a Urination being fully recovered in 2 1-3 days 60 Frequency and velocity of 1 urination turning normal .sup. 70.sup.b A great improvement on prostate 1 60~70.sup.c Fully recovering in 2 months 3 .sup.aPatients having urinary tract adhesions (atypical symptom after prostate cancer surgery) after laser surgery. Urinary tract adhesions will last several months, and a general drug thereof is α-blocker. Instead, the extract of Asplenium nidus L. of the preferable embodiment in the present invention fast response to affect parts, effectively improving symptoms of urinary tract adhesions. .sup.bA prostate cancer patient .sup.cPatients of benign prostatic hyperplasia

(46) In summary, the extract of Asplenium nidus L. in the preferable embodiment of the present invention has natural active substances against androgen, and therefore, the said extract of Asplenium nidus L. is sufficient to be used on the therapy of diseases caused by hormone imbalance, prostate diseases (including benign prostate hyperplasia, prostate cancer and prostatitis) in particular. Also, the said extract of Asplenium nidus L. will not lead to any side effects to patients. The said extract of Asplenium nidus L. is easily to obtain via a convenient extracting method, and which is capable of being further applied to therapy or prophylaxis of the prostate disease, as an active substance in medications or health products thereof, to improve males' prostate issues.

(47) Although the invention has been described in detail with reference to its presently preferable embodiments, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.