Active substance of <i>Morchella</i>, its use and a composition thereof for improving the reproductive function
11730783 · 2023-08-22
Assignee
Inventors
- Chin-Chu Chen (Taoyuan, TW)
- Szu-Yin Wu (Taoyuan, TW)
- Hsiao-Ling Chang (Taoyuan, TW)
- Yen-Po Chen (Taoyuan, TW)
Cpc classification
A61K9/19
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
International classification
A61K47/10
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
Abstract
The present invention provides an active substance of Morchella, its use and a composition thereof for improving disturbance of reproductive function, especially, for manufacturing a pharmaceutical composition to improve disturbance of reproductive function induced by obesity or metabolic syndrome. The composition with the active substance of Morchella can effectively improve the structural integrity of testicular tissues and sperm, increase testosterone levels in the blood, and reduce the oxidation stress in sperms.
Claims
1. A method for improving disturbance of reproductive function by administering a composition to a mammal in need, wherein the reproductive function of the mammal is improved as compared to a mammal not administered the composition; wherein the composition comprises an effective amount of an active substance of Morchella; wherein the Morchella comprises Morchella esculenta with a deposit number BCRC-36352, Morchella crassipes with a deposit number BCRC-36336, or a combination thereof.
2. The method of claim 1, wherein the active substance of Morchella is a lyophilized powder obtained after fermentation and freeze-drying of mycelia of Morchella.
3. The method of claim 1, wherein the active substance of Morchella is an extract obtained by extracting mycelia of Morchella with hot water or ethanol.
4. The method of claim 1, wherein the reproductive function comprises a testosterone level, sperm type, oxidative stress in sperms, the structural integrity of testicular tissues, or a combination thereof, wherein the reproductive function of the mammal is improved as compared to a mammal not administered the composition.
5. The method of claim 4, wherein the improving disturbance of reproductive function is increasing 2 to 2.5 times of testosterone levels in the blood as compared to a mammal not administered the composition.
6. The method of claim 4, wherein the improving disturbance of reproductive function is reducing the proportion of abnormal sperms with broken tails, bent tails, or bent necks as compared to a mammal not administered the composition.
7. The method of claim 4, wherein the improving disturbance of reproductive function is reducing 30 to 50% of reactive oxygen species produced in sperms to reduce the oxidative stress in sperms as compared to a mammal not administered the composition.
8. The method of claim 4, wherein the improving disturbance of reproductive function is reducing the number and volume of gaps within the luminal center of the seminiferous tubules or between the seminiferous tubules to improve the structural integrity of a testicular tissue as compared to a mammal not administered the composition.
9. The method of claim 1, wherein the disturbance of reproductive function is induced by obesity and/or metabolic syndrome.
10. The method of claim 9, wherein the metabolic syndrome is diabetes.
11. The method of claim 1, wherein the active substance of Morchella is prepared by the following methods: (a) inoculating Morchella mycelia on a medium for solid-state culture; (b) inoculating the Morchella mycelia cultured in step (a) in a liquid medium for liquid culture; (c) inoculating the Morchella mycelia cultured in step (b) in a fermenter for fermentation; and (d) extracting Morchella mycelia with hot water or ethanol to obtain an extract.
12. The method of claim 11, wherein a temperature of step (c) is 23 to 28° C., the ventilation is 0.5 to 1.0 vvm, the rotation speed is 30 to 50 rpm, and/or the number of cultivation day is 6 to 10 days.
13. The method of claim 1, the composition further comprises an additive selected from the group consisting of an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, and a combination thereof.
14. The method of claim 1, the composition is a medicine, feed, drink, nutritional supplement, dairy product, food, or health food.
15. The method of claim 1, the composition is in the form of powder, lozenge, granulation, microcapsule, ampoule, liquid spray, or suppository.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
DETAILED DESCRIPTION OF THE EMBODIMENTS
(5) Source of Fungus
(6) Morchella can be selected from the group consisting of M. angusticeps, M. conica, M. elate, M. deliciosa, M. esculenta, and M. crassipes. In a preferred embodiment, two Morchella species are purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute. One is Morchella esculenta, with a deposit number BCRC-36352, the other is Morchella crassipes, with a deposit number BCRC-36336. The active substances of Morchella of the present invention are not limited to the ones obtained by the above-mentioned species.
(7) Fungus Cultivation
(8) Mycelia of the purchased Morchella were inoculated on the solid medium to activate the fungus. In a preferred embodiment, the solid medium is Potato dextrose agar (PDA). After the growth of Morchella mycelia was complete, a cube of fresh mycelia together with the solid culture medium was inoculated into a flask containing 1 L of the liquid medium. In a preferred embodiment, the formulation of the liquid medium is shown in Table 1. In a preferred embodiment, the mycelia are liquid cultured under the conditions of a temperature of 25° C. and a speed of 50 to 150 rpm for 4 to 7 days to complete the growth of the mycelia.
(9) TABLE-US-00001 TABLE 1 Formulation of the liquid medium is shown as follows: Composition Proportion Sucrose 1-10% Yeast extract, YE 0.1-2% Soybean powder 1-10% KH.sub.2PO.sub.4 0.01-0.2% MgSO.sub.4 0.01-0.2%
(10) Subsequently, the mycelia growing completely in the flask were inoculated in a sterilized medium in a 100-liter fermenter for mass production. In a preferred embodiment, the Morchella mycelia are mass-cultured at a temperature of 25° C., a ventilation of 0.5-1.0 vvm, and a rotational speed of 40 rpm for 6 to 10 days. When the dry weight of the mycelia stops increasing or when the sucrose residue was less than 1000 mg/L, the fermenter was heated to stop cultivation. In a preferred embodiment, the fermenter was heated to 100° C. for 15 minutes to stop cultivation.
(11) Preparation of Lyophilized Powder
(12) After fermentation was completed, a part of the whole fermented liquid (containing the culture medium and the mycelia) in the fermenter was spread on a plate and freeze-dried to prepare a “lyophilized powder of whole fermented liquid.” In a preferred embodiment, the freeze-drying temperature is set at 30° C. and the freeze-drying duration is at least 3 days. The other part of the whole fermented liquid was centrifuged, and the Morchella mycelia were freeze-dried to obtain “lyophilized powder of mycelia.” In a preferred embodiment, the whole fermented liquid was centrifuged at 4500 rpm by using a continuously centrifugal decanter centrifuge.
(13) Preparation of Extract
(14) An appropriate amount of “lyophilized powder of whole fermented liquid” was resuspended in pure water. In a preferred embodiment, the volume of pure water for resuspending is 5 to 40 times, more preferably 20 times, of the weight of the lyophilized powder. Subsequently, the mixture was heated at 121° C. for 20 minutes, and then the mycelia were removed by centrifugation (4500 rpm, using a continuously centrifugal decanter centrifuge) to obtain an extract of whole fermented liquid. The extract of whole fermented liquid was lyophilized to obtain “hot water extract.” An appropriate amount of “lyophilized powder of mycelia” was resuspended with alcohol. In a preferred embodiment, the volume of the alcohol for resuspending is 5-40 times, more preferably 20 times, of the weight of the lyophilized powder. In a preferred embodiment, the concentration of alcohol is 95-100%. In a preferred embodiment, the alcohol is ethanol.
(15) The resuspended lyophilized powder of mycelia was sonicated for 2 hours and then centrifuged (4500 rpm, using a continuously centrifugal decanter centrifuge) to obtain mycelium extraction. The mycelium extraction was concentrated under reduced pressure to obtain “ethanol extract.” The hot water extract and the ethanol extract were thoroughly mixed to obtain an “extract mixture” as an active substance for Morchella feeding tests. In a preferred embodiment, the hot water extract and the ethanol extract are mixed in a ratio of 1:1 by weight.
(16) Test Animals
(17) Male 5-week old Sprague Dawley (SD) rats were purchased from BioLASCO Taiwan Co., Ltd. The transparent cages for the experimental rats were sterilized with 75% ethanol before the rats were put in. Two rats were housed in a cage at a temperature in the range of 23±1° C., with humidity maintained at 40-60%, light and dark cycles of 12/12 (7:00 a.m. to 6:59 p.m. bright, and 7:00 p.m. to 6:59 a.m. dark). Feed pellets (Laboratory Rodent Diet 5001, PMI® LabDiet®, St. Louis, Mo., U.S.A.), feed, and distilled water were supplied ad libitum.
(18) Animal Feed
(19) Feed powder Laboratory Rodent Diet 5001M (PMI Nutrition International, Inc., USA) was purchased from Young Li Co., and lard is selected as fat and was purchased from High Science Co., Ltd., (Taiwan). The formulation is shown as follows:
(20) TABLE-US-00002 Weight percentage (%) Control Group High-Fat Diet Group Crude protein 23.9 21.5 Crude fat 5.0 18.4 Carbohydrates 53.8 40.8 Ash 7.0 6.0 Others 10.3 13.3
(21) The feed of the high-fat diet group was made by adding lard to the feed powder, wherein the fat was 40% of total calories, to induce insulin resistance.
(22) Test Groups
(23) Male 5-week old Sprague-Dawley rats were housed for one week, with 8 in each group, for a total of 32 animals in four groups. One group was the control group while the other three groups were feed with the above animal feed and injected with streptozocin (STZ) to create diabetes model rats. Out of three diabetic groups, two groups were administrated with Morchella esculenta and Morchella crassipes, respectively.
(24) Name and abbreviation of each group are shown as follows:
(25) 1. Control group (C)
(26) 2. Diabetes group HFD/STZ-Diabetes (D)
(27) 3. Diabetes group feeding Morchella esculenta (D+ME)
(28) 4. Diabetes group feeding Morchella crassipes (D+MC)
(29) Tube Feeding and Sacrifice
(30) After diabetes was successfully induced in the experimental rats, the rats were fed with samples by Tube Feeding. The extract mixture was dissolved in a carrier solution and fed with an amount of 500 mg/kg body weight (B.W.) to the Morchella esculenta (D+ME) diabetic group and Morchella crassipes (D+MC) diabetic group. The control group was administered the same volume of vehicle solution (vehicle, 0.1 mol/L citric acid buffer, pH 4.5) as the volume administered to the experimental groups. Body weights of the experimental animals were measured and recorded weekly. After four weeks of sample feeding, the experimental animals underwent Oral Glucose Tolerance Test (OGTT). After animals of each group were fasted for 12 hours, blood was collected from tail vein, and concentration of blood glucose was measured. At the end of the experiment, carbon dioxide was used for anesthesia of the rats, syringe and centrifuge tube were pre-wetted with heparin (500 IU/mL), and blood was drawn from the abdominal aorta to sacrifice the rats. The obtained blood was taken as whole blood, loaded into a 15 mL centrifuge tube, and centrifuged at 3000×g for 15 minutes at 4° C. After centrifugation, the supernatant was collected as rat plasma. At the same time, the rats were sacrificed and the organs were removed and weighed. Liver, testis, and hypothalamus were stored at −80° C. for further experimental analyses, while sperms in epididymis were analyzed on the day of sacrifice.
(31) Sections and Staining of Testis
(32) Rats were sacrificed, and their testes were immersed in 10% formaldehyde (formalin) for later use. The testis tissues fixed with formaldehyde were cut with a scalpel to obtain a thickness of 0.5 cm tissue. Afterward, the tissues were immersed in formaldehyde again. Stained sections of the testis tissues were prepared by hematoxylin and eosin (H&E) staining. In hematoxylin and eosin (H&E) staining, hematoxylin is a basic dye for staining nucleus chromatin and cytoplasmic ribosomes a purplish blue, and eosin is an acid dye for staining the composition of the cytoplasm and extracellular matrix red. Therefore, based on the staining results, the structure of the tissues can be determined. After the sections of rat testis tissues were stained, the type of the seminiferous tubules of the testes was observed.
(33) Collection of Sperms
(34) Sperms in the epididymis were collected using a swim-up method. The epididymis was removed from the sacrificed rats and placed in a beaker containing 8 mL of RPMI culture medium. Epididymis was cut in two sections with dissecting scissors, placed on a rocker to shake for 10 minutes, and centrifuged at 190×g for 5 minutes. After centrifugation, the samples were placed in an incubator with 5% carbon dioxide at 37° C. for 30 minutes. Finally, sperms with better activity in the upper layer were collected to observe the abnormalities of the sperms.
(35) Determination of Testosterone Levels
(36) Concentrations of testosterone in the serum of the plasma samples were tested by testosterone ELISA kits. 50 μL of rat plasma were added to 50 μL of acetylcholinesterase (AChE) and anti-testosterone plasma for reaction. Next, the supernatant was removed and washed 5 times with a wash buffer. Ellman's reagent was added as AChE substrate and shaken in the dark for 80 minutes to complete the test sample. The absorbance of the sample at 412 nm was measured with an ELISA reader, and the concentration of testosterone (ng/mL) in the serum was obtained by converting the standard curve of the standard into the absorbance of the sample.
(37) ROS Assay
(38) DCFH-DA is a fluorescent probe that does not itself fluoresce. When DCFH-DA enters a cell, hydrolysis by esterase generates a non-fluorescent polar substance, DCFH, which is oxidized by intracellular ROS after it enters the cell, making it a fluorescent DCF, which can indirectly measure the amount of intracellular ROS production.
(39) DCFH-DA was added to sperm RPMI solution containing 1×10.sup.6 sperms/mL by adjusting the obtained 1 mL sperm of each group to a final concentration of 20 μM and incubated at 37° C. for 30 minutes. Then, the samples were centrifuged at 760×g for 5 minutes. The supernatant was removed after centrifugation and washed once with PBS. The samples were centrifuged again to remove supernatant, and 1 mL of PBS was added to resuspend the cells. The samples were analyzed by flow cytometry, which uses cell quest software. Ten thousand (10,000) cells were collected as a test sample, and the fluorescence intensity emitted by the machine was used to estimate the amount of ROS in the cells of the test samples. A high absorbance indicates a high level of ROS in the cells of the samples.
(40) Statistical Analysis
(41) Statistical analysis of the data was conducted using Statistical Product & Service Solutions (SPSS) software version 19.0, and the experimental results are expressed as Mean±S.E.M. Line charts were plotted using a Paired-Sample T-test with a statistical significance of p<0.01 (**) and p<0.05 (*). Histograms were plotted based on One-way analysis of variance One-way ANOVA and then using Duncan's Test for multiple comparison methods, with p<0.05 representing statistically significant differences.
(42) Based on the experimental results obtained from the above experiments, the details are described as follows.
(43) The Integrity of Testicular Tissues
(44) Staining results of rat testis tissues are shown in
(45) In contrast, gaps within the luminal center of the seminiferous tubules or between the seminiferous tubules of the diabetes groups feeding Morchella esculenta (D+ME) and Morchella crassipes (D+MC) are significantly fewer when compared to the diabetic group (D), indicating that these groups have more sertoli cells and Leydig cells. Based on the results of the stained sections of the testicular tissues, the decreased number of sertoli cells and Leydig cells in the testis caused by diabetes can be restored back to normal status by administration of the active substance of Morchella, suggesting that Morchella has a beneficial effect on the male reproductive function.
(46) Type of Sperms
(47) The collected sperms are shown in
(48) In contrast, sperm in diabetes groups feeding Morchella esculenta (D+ME) and Morchella crassipes (D+MC) have similar structures to those in the control group (C), indicating that administrating the active substances of Morchella can reduce structural damages on sperms caused by diabetes.
(49) Testosterone Levels
(50) The results of the testosterone levels in rat plasma samples are shown in
(51) In contrast, the testosterone levels of two diabetes groups feeding Morchella esculenta (D+ME) and Morchella crassipes (D+MC) is about 1.0 ng/mL and 1.25 ng/mL, respectively, which are both higher than that of the diabetic group (D). The testosterone levels in both D+ME and D+MC groups were significantly higher when compared to that of the control group (C). Based on these results, it is shown that administration of active substances of Morchella can stimulate increases in the low secretion or production of testosterone caused by diabetes.
(52) ROS Content
(53) The results of ROS content in sperms are shown in
(54) In contrast, ROS content of diabetes groups feeding Morchella esculenta (D+ME) and Morchella crassipes (D+MC) decreased to about 120% and 100%, respectively, which are similar to the content of the control group (C). Based on these results, it can be concluded that administration of active substances of Morchella can reduce the oxidative stress caused by diabetes.
(55) Based on the above experimental results, it was confirmed that the active substance extracted from Morchella can increase the reproductive function of male animals, suggesting a novel use of Morchella in the medical field is developed. Therefore, a composition comprising the active substance of Morchella can be manufactured and administered to an individual in an effective amount to achieve a healing effect in the reproductive function.
(56) As used herein, an “effective amount” refers to an amount that is sufficient to produce the aforementioned prophylactic and/or therapeutic effect. Based on in vitro cell culture experiments, the aforementioned effective amount is defined as “μg/ml” based on the total volume of cell culture medium used in each culture. Based on animal model experiments, the aforementioned effective amount is defined as “g/60 kg body weight/day.” In addition, the data of effective amount obtained via in vitro cell culture experiments can be converted to a reasonable effective amount for animal use by the following formula:
(57) 1. In general (Reagan-Shaw et al., 2008), 1 “μg/ml” units (based on the effective amount of in vitro cell culture experiments) may be equivalent to 1 “mg/kg body weight/day” units (based on the effective amount of rat model experiments), and, based on that metabolic rate of a rat is six times of that of a human, the effective human dose can be found.
(58) 2. Therefore, an effective amount for use in mice based on an in vitro cell culture experiment of 500 μg/ml is calculated as 500 mg/kg body weight/day (i.e., 0.5 g/kg body weight/day). Further, taking into account the differences in the aforementioned metabolic rates, an effective amount for human use may be taken as 5 g/60 kg body weight/day.
(59) 3. Based on the test results reported above, a validated dose based on a rat experiment is 500 mg/kg body weight/day and, therefore, a reasonably effective dose for human use should be 5 g/60 kg body weight/day.
(60) In a preferred embodiment, an effective amount of the active substance of Morchella contained in the composition is 500 mg/60 kg to 10 g/60 kg body weight/day.
(61) The composition further comprises an additive. In a preferred embodiment, the additive may be an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a combination thereof. The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, or a combination thereof. This preservative, such as benzyl alcohol, parabens, extends the shelf life of pharmaceutical compositions. The diluent can be selected from water, ethanol, propylene glycol, glycerol, or a combination thereof. The filler can be selected from lactose, nougat, ethylene glycol of high molecular weight, or a combination thereof. Absorption enhancers may be selected from dimethylsulfoxide (DMSO), laurocapram, propylene glycol, glycerol, polyethylene glycol, or a combination thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame, or a combination thereof. In addition to the additives listed above, other additives that are suitable for use may be selected based on requirements without affecting the medical effect of the active substance of Morchella.
(62) The composition can be developed as a different product in the medical field. In a preferred embodiment, the composition is a pharmaceutical product, feed, beverage, nutritional supplement, dairy product, food, or health food.
(63) The composition may take different forms depending on the needs of the receiver. In a preferred embodiment, the composition is in the form of powder, lozenge, granulation, microcapsule, ampoule/ampule, liquid spray, or suppository.
(64) The composition of the invention can be used in animals or humans. Without affecting the effect of the active substance of Morchella, the composition comprising the active substance of Morchella can be made into any pharmaceutical form and administered to the animal or human in a suitable manner depending on the type of the drug.
(65) Preparation of the Composition
(66) Composition 1: The hot water extract (20 wt %) was taken as the active substance of Morchella, mixed well with benzyl alcohol (8 wt %) as a preservative, glycerin (7 wt %) as a diluent, and dissolved in purified water (65 wt %) to produce a pharmaceutical composition of the present invention in liquid form. The aforementioned wt % means the ratio of each ingredient to the total weight of the composition. Store at 4° C. for later use.
(67) Composition 2: The ethanol extract (15 wt %) was used as the active substance of Morchella, mixed well with benzyl alcohol (5 wt %) as preservative, glycerin (10 wt %) as a diluent, and dissolved in purified water (70 wt %) to produce a pharmaceutical composition of the present invention in liquid form. The aforementioned wt % means the ratio of each ingredient to the total weight of the composition. Store at 4° C. for later use.