Myomin: natural aromatase inhibitor for estrogen-related conditions.Introduction
Aromatase inhibition is considered a novel methodology in the treatment of estrogen-responsive diseases. Aromatase inhibitor (Al) drugs have shown very promising results in clinical trials. In the Breast International Group 1-98 study, adjuvant treatment with the Al letrozole was found to reduce the risk of recurrent endocrine-responsive breast cancer in postmenopausal women. (1) This result was reinforced by the MA-17 trial, which found that extended adjuvant therapy with the same Al, up to 48 months, provides greater benefit. (2)
The success of the Als on breast cancer and their potential effect on other estrogen-responsive diseases have led our team of researchers to explore possible alternative Als in herbs. What we discovered was that the herbal combination of Curcuma, Cyperus, and Astragalus exhibits aromatase inhibition activity in rats, as presented in the following sections. This combination of herbs will be referred to as Myomin in this article. The succeeding in vitro, in vivo, and clinical studies will demonstrate Myomin's mechanism, functions, and applications.
MATERIALS AND METHODS 1. Culture of Primary Ovarian Granulosa Cells in Vitro
1.1 Obtaining and Culture of Ovarian Granulosa Cells
Bilateral ovaries were obtained aseptically from 21- to 23-day-old female SD rats that had each been injected with 0.5 mg diethylstilbestrol per rat for three days. (3) Excess fat and connective tissues were trimmed from the ovaries in surgery. Then the ovaries were punctured to release the granulosa cells in McCoy's 5A media. Through centrifugation for 7 minutes at 1000 rpm, cells were recovered and then eluted 3 times with culture media. The precipitate was then washed and suspended in the required McCoy's 5A media. After cell counting, the cells were inoculated in the 96 well plates to a final concentration of 3-6 x 104 cells per well and placed in the tissue culture incubator at 37[degrees]C, in a 95% air/5% [CO.sub.2] atmosphere.
1.2 Effect of Myomin on the Survival Rate of the Ovarian Granulosa Cells
Twenty-four hours after inoculation, the cells were transferred to McCoy's 5A culture media containing 5, 10, 20, 40 and 80 [micro]g/ml Myomin, respectively, and a McCoy's 5A culture medium containing 0.05% absolute alcohol. (4) Cells of 6 to 9 wells were treated as a group at the same dose. After 24, 48 and 72 hours' treatment, 10 [micro]l Cell Count Kit (CCK-8) solution was added in every well. (CCK-8 is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. Since the CCK-8 solution is very stable and has little cytotoxicity, a longer incubation, such as 24 to 48 hours, is possible). The plates were then placed in the tissue culture incubator at 37[degrees]C, in a 95% air/5% [CO.sub.2] atmosphere for 1 to 4 hours. The absorbance of every well was measured with ELISAat 450 nm. This was repeated 3 times and the mean calculated. Cells of the control group were considered as 100%. Survival rate was calculated according to the following equation: survival rate = the mean absorbance of the experimental group/ the mean absorbance of the control group x 100%. Under microscopic examination, living cells had been stained yellow and dead cells appeared to have no stains with an increased volume. (5)
2. Effect of Myomin on the Expression of Aromatase in Ovarian Granulosa Cells
2.1 Expression of Aromatase in Ovarian Granulosa Cells
Twenty-four hours after the growth of cells on slides, the experimental cultures were transferred to McCoy's 5A culture media containing 0.01 lU/ml follicle stimulating hormone (FSH) and 0.5 [micro]M and rostenedione for a 72-hour culture. At the same time, the control cultures were transferred to a fresh McCoy's 5A culture medium for a 72-hour culture and fixed in a formic acid-glacial acetic acid solution (3:1 by volume). (6-8) These were incubated with 0.03% [H.sub.2][O.sub.2] in PBS for 10 minutes and washed with distilled water. These were incubated again in PBS for 5 minutes and blocked with normal goat blood serum for 10 minutes at room temperature, then incubated with the primary antibody (rabbit anti-rat P450 aromatase IgGs overnight at 4[degrees]C). After incubation, these were washed with PBS 3 times, incubated with the secondary antibody (biotinylated anti-rabbit IgGs) for 10 minutes at room temperature, washed with PBS again, and colored with DAB. After being washed with water, the cultures were counterstained with hematoxylin and mounted. The PBS-treated cultures were considered as negative control.
2.2 Effect of Myomin on the Expression of Aromatase in Ovarian Granulosa Cells
Twenty-four hours after the growth of cells on slides, the experimental cultures were transferred to McCoy's 5A culture media containing 0.01 lU/ml FSH, 0.5 [micro]M androstenedione, and 30 [micro]g/ml Myomin for a 72-hour culture. At the same time, the control cultures were transferred to a fresh McCoy's 5A culture medium containing 0.01 lU/ml FSH and 0.5 [micro]M androstenedione for a 72-hour culture and fixed in a solution of formic acid-glacial acetic acid (3:1 by volume). These were incubated with 0.03% [H.sub.2][O.sub.2] in PBS for 10 minutes and washed with distilled water. These were incubated in PBS again for 5 minutes and blocked with normal goat blood serum for 10 minutes at room temperature and incubated with the primary antibody (rabbit anti-rat P450 aromatase IgGs) overnight at 4[degrees]C. After incubation, the cultures were washed with PBS, incubated with the secondary antibody (biotinylated anti-rabbit IgGs) for 10 minutes at room temperature, and colored with DAB. After washing with water, the cultures were counterstained with hematoxylin and mounted. PBS-treated cultures were considered as negative control. Positive results: particles in the positive cells appeared brown, and in negative cells no stains were employed. The percentage of living cells in all plates was calculated and the data analyzed with statistical analysis system software.
3. An in Vivo Experimental Model of Endometriosis
Establishment of the Rat Model of Endometriosis Induced by Transplanting Uterus Fragments to the Surface of the Peritoneum (Autotransplantation).
The model is described as follows9: The rats were anesthetized intraperitoneally with 3% pentobarbital (with a dose of 30 mg/kg) and arranged supine on an operation table. Surgery was carried out following aseptic precautions. The abdomen was shaved and cleaned with 95% ethyl alcohol. To expose the uterus, a midline incision was made and then extended to 3 cm from 0.5 cm above the pubic symphysis. From the distal end of the cornu uteri, a 1 -cm segment was cut and placed in a warm sterile saline solution. Both remaining ends were immediately ligated, leaving the ovaries and oviducts. The excised endometrium segment was separated from the myometrium, split longitudinally, and cut into four 5 mm x 4mm pieces. These were placed in RPMI-1640 media at 37[degrees]C until ready to be used. Each 5 mm x 4 mm piece was sewn to the abdominal membrane with the membrana mucosa facing the abdominal cavity surface using alete nylon suture. After three weeks, the belly was cut open again. Growth of the ectopic endometrium was observed and the cyst measured with slide gage (length x width x height, [mm.sup.3]).
The Grouping and Dosage Regimen of the Management of Endometriosis in Rats
After the second surgery, the successful model rats were divided into two groups randomly, six rats per group:
* positive control group: equal volume 0.5% CMC solution per day, per rat by intragastric administration, for 28 days consecutively;
* Myomin group: intragastric administration for 28 days consecutively at a dose of 100 mg/kg Myomin per day per rat.
Twenty-one days after intragastric administration, the rats in both groups were anesthetized. Surgery was again performed to obtain the ovaries, ectopic endometrium, and remaining ectopic uterus. These were placed in formaldehyde for the following histological study.
After the conventional fixation, anhydration, embeddings, and sectioning, the ovary and ectopic endometrium samples were deparaffinaged in dimethylbenzene and dehydrated in gradient alcohol. Treatment with 0.03% [H.sub.2][O.sub.2] in deionized water for 5 to 10 minutes followed in order to block the endogenous peroxidase. These were washed with distilled water and incubated in PBS for 5 minutes. These were incubated with solution A (blue) for 10 to 15 minutes at room temperature. Then the solution was discarded. With the samples unwashed, these were incubated with the primary antibody (rabbit anti-rat P450 aromatase IgGs, 1:100 diluted) for 2 to 3 hours at 37[degrees]C and washed with PBS, 3 times for 3 minutes per washing. These were then treated with solution B (yellow) for 10 to 15 minutes at room temperature or 37[degrees]C and washed 3 times with PBS for 3 minutes per washing. Following that, the samples were treated with solution C (orange) for 10 to 15 minutes at room temperature or 37[degrees]C and washed with PBS 3 times for 3 minutes per washing. These were colored with DAB, washed with water, counterstained with hematoxylin, and mounted. The stained sections were observed by microscopy, and digital images were collected. The mean grayscale values of the images were obtained through the MIVNT image analysis system.
1.1. Cultured Primary Ovarian Granulosa Cells
About 4 x [10.sup.5] cells were gained from every ovary. One day after inoculation, ovarian granulosa cells adhered well and spread out. Feelers presented confluence, with most conglomerately and a few singly. A lipid droplet was inside each cell. Cell appearance was just as the previous papers reported. The cells can survive in McCoy's 5A media for at least 7 days.
1.2. Effect of Myomin on the Survival Rate of the Cells
This particular experiment was performed to determine the appropriate safe dosage of Myomin to be used in tissue cultures for ovarian granulosa cell growth. Twenty-four hours after subculturing, the cells were transferred to the McCoy's 5A media containing different dosages of Myomin; those transferred to the McCoy's 5A media containing 0.05% alcohol served as control. Forty-eight hours after the previous step, the cells were counted. The result of this experiment shows that doses of up to 20 [micro]g/ml Myomin did not change the quantity of living cells, while concentrations of at least 40 [micro]g/ml Myomin decreased the quantity significantly. By extrapolation, the ideal Myomin dose for succeeding experiments on ovarian and endometrial tissues is 30 [micro]g/ml.
2.1. Enhancement of FSH on Aromatase Expression
Twenty-four hours after the growth of cells on the slides, the experimental cultures were transferred to McCoy's 5A culture media containing 0.01 lU/ml FSH and 0.5 [micro]M androstenedione for a 72-hour culture. The cells were observed in situ. Immunohistochemical staining showed that the enchylema of most cells treated with only media-containing FSH had been stained yellow under microscopic examination. In the cells of the negative control group, no stains were employed.
2.2 Myomin Reduced Aromatase Expression in FSH-Stimulated Ovarian Granulosa Cells
Twenty-four hours after the growth of cells on the slides, the experimental cultures were transferred to McCoy's 5A culture media containing 0.01 IU/ ml FSH, 0.5[micro] androstenedione, and 30 [micro]g/ml Myomin for a 72-hour culture. The cells were observed in situ. Under microscopic examination, in the cells of the negative control group, no stains had been employed (Figure 1A, p. 72). In the untreated positive control group, stained cells accounted for 94.0 [+ or -] 45.0% of the total cells (Figure 1B). Stained cells decreased by adding Myomin into the culture media and accounted for 31.0 [+ or -] 11.0% of the total cells (Figure 1C). Using statistical t-analysis, a significant difference existed between the two groups (p < 0.05).
[FIGURE 1 OMITTED]
3. The Effect of Myomin on the Expression of Aromatase in the Rat Ovary and Rat Ectopic Endometrium in Vivo
Myomin Reduced Aromatase Expression in the Ectopic Endometrium of Rats in Vivo
Aromatase was expressed markedly in the ectopic endometrium of the positive control group. Arornatase expression significantly decreased in the ectopic endometrium of the Myomin group 28 days after therapy in comparison with those in the ectopic endometrium of the positive control group (Figures 2A to 2C).
[FIGURE 2 OMITTED]
Using the image analysis system, the average grayscale value of aromatase expression in the positive control group was 108.9 [+ or -] 29.03, and that in the Myomin group was 34.7 [+ or -] 5.2 (see Table 1, p. 71), a significant difference between the two groups.
Table 1: Aromatase Expression in Ovarian and Ectopic Endometrium Tissues Measured through Grayscale Values Group Ectopic endometrium Ovary (Mean [+ or -] (Mean [+ or -] SD) SD) PBS negative control group 48.0 [+ or -]8.50 29.7 [+ or -] 6.40 Positive control group 108.9 [+ or -] 29.03 149.8 [+ or -] 33.86 Myomin group 34.7 [+ or -] 5.20 47.0 [+ or -] 9.19 * Significant difference compared with positive control group (p < 0.5)
Myomin Reduced Aromatase Expression in the Ovary of Rats in Vivo
Aromatase was expressed markedly in the ovaries of the positive control group. After 28 days of therapy, aromatase expression was significantly lower in the ovaries of the Myomin group than in those of the positive control group (Figures 3A--3C). Using the image analysis system, the average grayscale value of aromatase expression in the ovaries of the positive control group was obtained at 149.8 [+ or -] 33.86, and that in the Myomin group was 47.0 [+ or -] 9.19 (Table 1).
[FIGURE 3 OMITTED]
4. Clinical Studies on Myomin
Myomin Reduces Estradiol in Postmenopausal Women
A clinical study involved 60 postmenopausal women with cysts and/ or fibroids (Figure 4). Postmenopausal women were studied because their estradiol levels are more stable than those of premenopausal women. Estradiol level in postmenopausal women is normally under 32 pg/ml. Initially, the average estradiol level of the women in the study was 74.52 pg/ml. After only 10 days on Myomin, their average estradiol level reduced to 38.84 pg/ml. The fibroids, cysts, and associated pain gradually reduced as well.
Figure 4. Effect Myomin on Estradiol Level of 60 Postmenopausal Women with Fibroids and Cysts Esstradiol level (pg/ml) Initial 74.52 After 10 days 38.84 Note: Table made from bar graph.
[FIGURE 4 OMITTED]
Myomin Effective for Fibrocystic Breasts
Another study involved 50 cases of fibrocystic breasts (Figure 5). There were 45 women in the study, ages 18 to 52 years old, and 5 men, ages 62 to 64 years old. Sixty-five percent of the women were under 35 years old. After 6 months, Myomin was effective in 60% of the patients, while improvement was seen in 30%. Only 5 cases did not notice any change in their condition. Among the men in the study, the cysts cleared completely in 3 cases. In 48 out of 50 cases, associated pain cleared or tremendously reduced.
Figure 5 Myomin on 50 Patients with Fibrocystic Breasts (6 months) Effective rate Effective 60% Improved 30% No Effect 10% Note: Table made from bar graph.
[FIGURE 5 OMITTED]
Myomin for Ovarian Cysts and Endometriosis
A 1994 clinical study was conducted of 255 patients with ovarian cysts and endometriosis (Figure 6). After 5 months, Myomin was effective in 147 patients (57.6%), while some improvement was observed in 87 patients (34.1%). No effect was observed in only 21 patients.
Figure 6. Myomin on 255 Patients with Overian Cysts and Endometriosis (5 months) Effective rate (%) Effective 57.6 Improved 34.1 NoEffect 8.2 Note: Table made from bar graph.
[FIGURE 6 OMITTED]
Myomin for Uterine Fibroids
A 2000 study was performed of 60 patients with uterine fibroids over 2.5 [cm.sup.3] in size (Figure 7). The patients were given Myomin with two other supplements: Angiostop, which inhibits angiogenesis, and Revivin, which helps to control abnormal growth. Both supplements are recommended in addition to Myomin for those with large uterine fibroids, among other conditions, especially those who experience bleeding and other related symptoms. Results of the study revealed that the combination is 69.2% effective on uterine fibroids after 6 months. Improvement was observed in 19% of the patients.
Figure 7.Myomin, Revivin and Angiostop on 60 Patients with Uterine Fibroids (6 months) Effective rate (%) Effective 69.2 Improved 19 No Effect 11.8 Note: Table made from bar graph.
[FIGURE 7 OMITTED]
Estrogen dictates important mechanisms such as protein production and cell division, and is vital to growth processes not just during puberty, but in adult life. However, its role in signaling cells to divide and multiply can trigger and promote abnormal growth of estrogen-responsive tissues. Minimizing estrogen, therefore, is a clear goal for estrogen-responsive conditions.
Aromatase is a key enzyme in the synthesis of estrogen. It converts androstenedione into estrone and testosterone into estradiol (Figure 8). Aromatase's important role in the production of estrogen has made it a focus in the search for viable therapies for estrogen-mediated conditions. Increased aromatase expression promotes estrogen production, especially at the site of hormone-responsive tumors such as in breast, endometrial, and ovarian cancer. (10) The development of several Al drugs, primarily for breast cancer, is based on this premise. The success of Als on this disease opens other potential applications. In fact, Als are also being explored as possible treatment for estrogen-responsive conditions such as endometriosis and prostate cancer. (11), (12)
[FIGURE 8 OMITTED]
Both in vitro and in vivo studies demonstrate that Myomin could be a potentially viable herbal Al. In the in vitro study in section 2.2, FSH was used to stimulate aromatase expression in cells. In the untreated positive control group, aromatase expression was signified by stained cells, which accounted for 94% of the total cells. After adding Myomin into the tissue culture, stained cells reduced to only 31% of the total cells, signifying a 67.02% reduction of aromatase expression.
The in vivo studies further reinforce the aromatase inhibition activity of Myomin (section 3). Aromatase expression was measured using grayscale values obtained through image analysis. In the ectopic endometrium and ovarian tissue positive control groups, the average aromatase expression was measured at 108.9 and 149.8, respectively. Aromatase expression is obviously much higher in these groups compared with that of the Myomin group, which has grayscale values of 34.7 for the ectopic endometrium and 47.0 for the ovarian tissue. This means that after administration of Myomin, aromatase expression in the ovarian tissue is almost comparable with that of the negative control group, while aromatase expression in the ectopic endometrium of the Myomin group is even much lower than the corresponding negative control group's value.
The clinical studies on Myomin demonstrate its efficacy in reducing estradiol and improving estrogen-responsive conditions such as fibrocystic breasts, uterine fibroids, ovarian cysts, and endometriosis (section 4). One particular case demonstrates how Myomin is beneficial for an estrogen-responsive condition like fibrous breasts. J. Blair, ND, of New Jersey has a 48-year-old female patient with a long history of dense breasts since she started menstruation more than 30 years ago. A mammogram was not adequate to test her breasts for growths; an ultrasound was always needed for further testing. The patient took Myomin for a year and a half. She reports that, for the first time in her life, a mammogram shows a clear picture: no more denseness at all. An ultrasound was no longer needed.
Myomin's estrogen-reducing effect is illustrated in a case report from J. lannetta, DC, of Maine, of a 57-year-old postmenopausal female patient who initially had very high estradiol at 413 pg/ml. After taking Myomin for 14 months, her estradiol reduced to 43 pg/ml. In a similar case, J. Wright, MD, of Washington has a male patient in his 50s who had elevated estradiol. None of the supplements he tried seem to have any effect on it. After taking Myomin for 4 months, his estradiol has sharply dropped. This particular case shows that Als like Myomin may have uses for estrogen-related conditions in men.
Indeed, as we learn more about the etiology of prostatic diseases, the importance of Als in men becomes increasingly clear. As men age, testosterone is more readily converted to estrogen through aromatase. (13) Some studies show that increased estrogen level is seen in problems such as benign prostatic hyperplasia (BPH), (14-16) prostate cancer, (17) prostatitis, (18), (19) and even gynecomastia. (20-22) In fact, some studies report that estrogen promotes the overgrowth of prostate tissue in BPH. (14-16) Evidence also suggests that local aromatase expression in the prostate increases in malignancy. The level of aromatase expression in malignant prostate tissue is actually comparable with that in breast cancer tissue. (17) As a result, local estrogen levels are also promoted at the prostate tumor site, increasing growth. Aromatase inhibitors will help block the action of aromatase, thereby minimizing the depletion of testosterone and reducing estrogen levels.
One case showed that enlargement of the prostate may well be estrogen-mediated. L. Bazakos, DC, of New York has a 55-year-old male patient with BPH. He took Myomin and Prosta Chi for over a year. A test showed that his prostate has reduced in size and is soft, almost like that of a 19-year-old boy. In another case, a 16-year-old male gynecomastia patient of B. Sanborn, DC, of Michigan responded well to Myomin. He had elevated levels of estradiol and dihydrotestosterone (DHT). After taking Myomin for two months, his estradiol level reduced from 300 to 70, while his DHT level reduced from 1100 to 250. A noticeable difference in the size of his breasts was also observed during this time.
Myomin has also been found to increase the 2/16[alpha] hydroxyestrone ratio (Figure 8), according to a case study from E. Schlabach, DC, of Ohio. A higher 2/16[alpha] hydroxyestrone ratio is associated with a decreased risk for estrogen-related neoplasms. Dr. Schlabach reported that, according to many patient urine laboratory tests, Myomin not only can reduce estradiol but also can quickly increase the 2/16[alpha] hydroxyestrone ratio. After a month of taking Myomin, the ratio increased to over 2.0 to 6.0.
Als like Myomin may also benefit those who are taking hormones such as progesterone, testosterone, androstenedione, or dehydroepiandrosterone (DHEA). These are precursors to estrone and estradiol (see Figure 8), both of which are potent forms of estrogen. Those taking hormone supplementation may accumulate estrone and estradiol if their production remains unchecked. Als help reduce the accumulation of these hormones. By inhibiting aromatase, synthesis of estrone and estradiol from androstenedione and testosterone is also blocked (see Figure 8), while production of the weaker form of estrogen, estriol, goes unhindered. Studies have shown that estriol provides the benefits that estrone and estradiol do and may even have a better safety profile. (23), (24) Those on bioidentical hormones may still have a risk for developing tumors or abnormal growths if they do not change their lifestyle. Exposure to xenoestrogens, which mimic the effect of estrogen, can increase their risk. Therefore, taking Myomin with the bioidentical hormones will provide a protective effect.
Aromatase inhibition may also play a role in reducing abdominal fat. Abdominal obesity has been associated with decreased testosterone levels and increased estradiol levels. Hypogonadism leads to the deposition of fat in the abdomen. As fat accumulates, aromatase activity increases at the site, causing more testosterone to be converted into estradiol. (25) An Al like Myomin blocks the action of aromatase, effectively preventing the testosterone from being converted into estradiol. This leads to more muscle mass and the loss of abdominal weight. Although more studies are needed to establish this concept, a case from E. Saugen, DC, of Minnesota demonstrates Myomin's effect on abdominal fat. A 48-year-old male patient was overweight, with much of his weight concentrated in the abdomen. After 6 months of taking Myomin, he lost 25 lbs. and 3 inches around the waist.
The studies presented here clearly demonstrate that Myomin inhibits aromatase expression in the in vivo ovary, in vivo ectopic endometrium, and in vitro cultured primary ovarian granulosa cells of rats, making it a potentially viable herbal Al. Clinical studies show that, through its aromatase inhibition function, Myomin is able to reduce estradiol and improve estrogen-responsive conditions like fibroids, fibrocystic breast, ovarian cysts, and endometriosis. While further investigation and more clinical studies are needed to identify its active ingredient and elucidate its complete mechanism, the studies and cases presented here provide compelling evidence that Myomin will be beneficial for estrogen-mediated conditions.
Tsu-Tsair Chi, NMD, PhD
P.O. Box 753
Atwood, California 92811
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by Tsu-Tsair Chi, NMD, PhD