# Patent

## Title
Mitochondria regulator composition

## Abstract

The present invention provides a mitochondria regulator composition, which comprises a wild bitter gourd extract. The mitochondria regulator composition can regulate the function of mitochondria in a cell, and further regulate the efficiency of adaptive thermogenesis and energy expenditure in a cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the priority of Taiwanese Patent Application No. 101129306, filed on Aug. 13, 2012, which is incorporated herewith by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a cell regulator, especially relates to a mitochondria regulator.
2. The Prior Arts
No matter at rest or at work, normal function of human organs and tissues must have adenosine triphosphate (abbreviated as ATP) involved. In cells about 90% of the ATP is generated by mitochondria, and oxygen is consumed when mitochondria produces ATP. It is estimated that 90% of the oxygen in the cell are used by mitochondria. Furthermore, when mitochondria metabolize three major nutrients (fatty acids, carbohydrates, and amino acids) efficiently, oxygen will be consumed no matter energy generated or heat generated. Therefore, mitochondria itself is high oxygen consuming organelle and mitochondria is the site with highest frequency of oxidation and reduction reactions in the cell.
In human cell, skeletal muscle use ATP most frequently. ATP required for muscle fiber contraction is dependent on vigorous oxidative phosphorylation reaction in mitochondria. Therefore, there are more blood vessels around the muscle fibers in order to provide enough oxygen and fuel molecules (i.e., fatty acids, carbohydrates, or amino acids).
In addition, fatty tissues in the body can be classified into two groups, namely white adipose tissue and brown adipose tissue, according to their mitochondria type, distribution site in the body and color. The white adipose tissue has smaller and less amount of mitochondria, and the fatty acids in the tissue serve as energy source during rest state and oxidative exercise. On the other hand, the brown adipose tissues are mainly distributed in the shoulder, neck and arm of hibernating animals, and have abundant of mitochondria. Because mitochondria contain many cell pigments, these pigments makes brown adipose tissue exhibits brown color.
Although oxidative metabolism in cellular mitochondria produces ATP, mitochondria in the brown adipose tissue are not designed to produce ATP as a mode of energy output. Especially, there are many thermogenin distributed on the inner membrane of mitochondria in the brown adipose tissue. The thermogenin is a protein that consumes all energy generated by oxidation of fuel molecules, thus no ATP can be generated. Instead, heat is produced and the energy is dissipated. Because the mitochondria in the brown adipose tissue are large and abundant, more heat is produced.
Therefore, brown adipose tissues rely on the unique heat generation system of mitochondria to oxidize fatty acids at low temperature or during ingestion. Without exercise, hibernating animals and newborn babies can also generate heat to adapt cold environment. Because the central temperature regulation system in the newborn babies is not mature, the heat production mode of brown adipose tissue is especially important to protect the newborn babies from tremble reaction. The thermogenin in the mitochondria of brown adipose tissue is an important factor to keep thermostasis.
The above thermogenin apply uncoupling reaction to reduce ATP generation in mitochondria, increase basic metabolism consumption and dissipate energy in heat form. Uncoupling protein (abbreviated as UCP) plays an important role as regulator in mitochondrial's uncoupling reaction. UCPs are proteins located in the inner membrane of mitochondria and there are three main expression forms, including UCP1, UCP2, and UCP3. UCP2 and UCP3 are mainly responsible for regulation of reactive oxygen species (ROS) synthesis. The expression levels of UCP2 and UCP3 are far less than that of UCP1 in mammals. UCP1, also called as “thermogenin”, is mainly expressed in brown adipose tissue and is mainly responsible for adaptive regulation of heat generation and heat consumption.
Peroxisome proliferator-activated receptor γ coactivator-1 (PGC1α), tfam (mitochondrial transcription factor A) and NrF1 (nuclear respiratory factor 1) are important transcription regulation factors in mitochondria biosynthesis, wherein PGC1α is a coactivator of PPARγ that mainly distributed in high oxygen consumption tissue, such as heart tissues or brown adipose tissues. PGC1α can regulate mitochondria biosynthesis and energy metabolism. Furthermore, tfam can bind to mitochondria DNA to regulate mitochondria transcription and replication, and NrF1 is also involved in regulation of mitochondria gene transcription. Prior studies have shown that increase of PGC1α□ concentration in the muscle can stimulate expression of abovementioned NrF and tfam of mitochondria genes, decrease possibility of obesity owing to aging, and decrease possibility of diabetes, and prolong life.
The material, bitter gourd, is a common traditional Chinese medicine. However, to the best of our knowledge, the bitter gourd has never been reported in regulation the mitochondria. The mitochondria regulator can modulate mitochondrial related proteins and genes effectively to enhance heat and energy generation efficiency. The mitochondria regulator composition of the present invention can be used as a healthy food composition or pharmaceutical composition for treatment of disease due to deficiency of thermogenin or mitochondria, mitochondrial dysfunction, mitochondria malfunction, or mitochondria dysregulation.
SUMMARY OF THE INVENTION
However, the prior studies of bitter gourd mainly focused on reduction of blood sugar, anti-oxidation activity, and regulation of immune reaction. The present invention discloses that the bitter gourd has significant ability to regulate biosynthesis of intracellular mitochondria and to improve production of energy and heat.
Therefore, the present invention provides a mitochondria regulator composition for regulating a mitochondria in a cell, which consisting essentially of a bitter gourd extract. The cells can be a white adipose tissue cell, a brown adipose tissue cell, or a muscle cell. One embodiment of the present invention, the bitter gourd extract of the present invention is used at least 5% of total diet.
In one example of the present invention, the mitochondria regulator composition can enhance the expression of UCP1 gene (SEQ ID NO:9), PGC1α gene (SEQ ID NO:4), or NrF1 gene (SEQ ID NO:12) in the white adipose tissue cell.
In another example of the present invention, the mitochondria regulator composition can enhance the expression of PGC1α (SEQ ID NO:4) or NrF1 (SEQ ID NO:12) in the brown adipose tissue cell.
In another example of the present invention, the mitochondria regulator composition can enhance the expression of PGC1α gene (SEQ ID NO:4) or tfam gene (SEQ ID NO:13) in the muscle cell.
The present invention also provides a method of regulating a mitochondria in a cell, comprising administering to a subject an effective amount of a bitter gourd extract, wherein the cell can be a white adipose tissue cell, a brown adipose tissue or a muscle cell. In one example of the present invention, the bitter gourd extract is a freeze-dry powder of whole bitter gourd fruit.
Wherein, the bitter gourd extract can enhance the expression of UCP1 (SEQ ID NO:9), PGC1α (SEQ ID NO:4), or NrF1 (SEQ ID NO:12) to regulate mitochondria activity in the white adipose tissue; can enhance the expression of can enhance PGC1α (SEQ ID NO:4), or NrF1 (SEQ ID NO:12) to regulate mitochondria activity in the brown adipose tissue; and can enhance the expression of PGC1α (SEQ ID NO:4) or tfam (SEQ ID NO:13) to regulate mitochondria activity in the muscle cell.
Using the bitter gourd extract of the present invention, the function of intracellular mitochondria can be regulated by enhancement of mitochondria biosynthesis and generation of heat, which further leading to regulation of heat generation and energy consumption efficiency.
The present invention is further explained in the following embodiment illustration and examples. Those examples below should not, however, be considered to limit the scope of the invention, it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and scope of the appended claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the oxygen consumption per gram body weight (VO2/gBW) of mice in 24 hours.
FIG. 1B shows a comparison of the area under curve of FIG. 1A.
FIG. 2A shows CO2 production per gram body weight (VCO2/gBW) of mice in 24 hours.
FIG. 2B shows a comparison of the area under curve of FIG. 2A.
FIG. 3A shows RQ value of mice in 24 hours.
FIG. 3B shows a comparison of the area under curve of FIG. 3A.
FIG. 4A shows gene expression in epididymal white adipose tissue (EWAT).
FIG. 4B shows gene expression in brown adipose tissue (BAT).
FIG. 4C shows gene expression in the muscle tissue.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention uses mice for study. Mice are divided into two groups, the control group and the experimental group. The diet of the experimental group contains 5% of wild bitter gourd powder, therefore, this group is also named as BGP group. The diet of the control group is a modification of AIN-93G basic diet, therefore, this group is also called Basal group.
Because increase of mitochondria biosynthesis and increase of heat and energy generation efficiency require consumption of oxygen, mice fed with experimental diet were monitored their oxygen consumption, CO2 production, and respiratory quotient at week 5. The oxygen consumption, CO2 production, and respiratory quotient of experimental group were compared with control group to examine if elevation trend was observed. If yes, further analysis of mRNA was conducted to determine if increase of mitochondria biosynthesis and increase of heat and energy generation efficiency contributed to elevation of oxygen consumption, CO2 production, and respiratory quotient. mRNA analysis was conducted by sampling of white adipose tissue, gastrocnemius, and brown adipose tissue of experimental group mice at 22 week for mRNA and protein analysis. If the BGP group mice fed with diet incorporating 5% wild bitter gourd showed significant elevation of fatty acid oxidation, adapted heat generation, and gene or protein related to mitochondria biosynthesis, the results provided evidences that wild bitter gourd can enhance mitochondria biosynthesis and increase efficiency of heat and energy generation. The above described methods are further explained in the following examples.
Data of the present invention are presented as means±SD. Differences between the two groups of mice were analyzed by Student's t test. Data was transformed into log or root square before statistic analysis if they are not normally distributed. P value <0.05 was considered significant.
Definition
The term “regulator” of the present invention is intended to mean capability to enhance or reduce an activity. Therefore, the mitochondria regulator composition of the present invention for use in regulation of mitochondria as described in the specification is intended to mean the use of the mitochondria regulator composition in direct contact with the mitochondria. In further examples, the mitochondria regulator composition of the present invention can be administered an effective amount to an individual when mitochondria require regulation to enhance or increase mitochondria activity.
Example 1
Diet Formulation of Bitter Gourd and Animal Study
Fresh wild bitter gourd (Cultivar Hualian No. 4) fruits were provided by Hualien District Agricultural Research and Extension Station of Taiwan. This cultivar was specifically bred based on a high PPAR activating activity. Whole fruits (included seeds) were washed, sliced, frozen and lyophilized. They were then grounded to produce the bitter gourd powder (BGP) sample and stored at −20° C. for making the BGP test diet.
Twelve eight-week old male C57BL/6J mice for animal studies were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Mice were acclimatized for 4 weeks and fed a non-purified diet (Rodent Chow, PMI Nutrition International, Brentwood, Mo. USA) before the experiment. After acclimation, mice were randomly assigned into two groups. The control group (n=6) was fed AIN-93 modified basal diet wherein carbohydrate sources were provided by 50% sucrose (abbreviated as basal group), while the experimental group (n=6) was fed AIN-93 modified basal diet by incorporating 5% (w/w) of a freeze dried powder of bitter gourd (abbreviated as BGP group). Before the experiment began, the experimental mice were acclimated for bitter taste for one week. All experimental mice were housed individually in stainless steel wire cages in an animal room with a 12 hour light and 12 hour dark cycle and constant temperature (22±2° C.). Throughout the acclimation and experimental period, mice had free access to water and diet. Diet was changed twice a week, and each mice body weight was recorded every week.
The diet formulations described above are shown in Table 1 below. The basal diet was a modification of AIN-93G diet, wherein the original carbohydrate source (62.95% of corn starch) was replaced by mixture of corn starch (Samayang genex, Korea) and sucrose in 12.95:50 ratio. The rest ingredients include casein (ICN, USA), cystine (Wako, Japan), cellulose (JRS, Germany), AIN-93G Vitamin Mix (ICN, USA), AIN-93 Mineral Mix (ICN, USA) and choline (Sigma, USA). To prepare diet, powder materials were mixed well. Then soybean oil (Taiwan Sugar) was added and mixed again. After sieving twice, diet was packaged in double layer sealing bag and stored at −20° C. until use. The BGP diet was formulated by incorporating 5% (w/w) of BGP into the basal diet by slightly adjusting the composition of casein, corn starch, soybean oil and cellulose based on the proximate composition of BGP, i.e., crude protein 4.5%, carbohydrate 54.6%, cellulose 38.2% and crude fat 2.7%.







TABLE 1







Diet Formulation of Basal Group and BGP Group containing 


Freeze Dried Powder of Hualien No. 4 Bitter Gourd









Composition %
Basal Group
BGP Group












Casein
20
19.775


L-Cystine
0.3
0.3


Corn Starch
12.95
10.22


Sucrose
50
50


Cellulose
5
3.09


Soybean Oil
7
6.865


AIN-93G Vitamin Mix
1
1


AIN-93G Mineral Mix
3.5
3.5


Choline
0.25
0.25


Freeze Dried Powder of Hualien 
—
5


No. 4 Bitter Gourd




Oligosaccharide
—
—


kcal/g
3.948
3.948


fiber/g
0.05
0.05


Energy of carbohydrate/total energy (%)
63.78
63.78


Energy of protein/total energy(%)
20.26
20.26


Lipid energy/total energy(%)
15.96
15.96





1. Diet of AIN-93 Vitamin Mix and AIN-93Mineral Mix are prepared according to formulation described in J. Nutt 123: 1939-1951 (1993) (Reeves et al., 1993).


2. Diet of BGP group was prepared incorporating bitter gourd powder into basal AIN-93 basal diet by slightly adjusting the compositions.






Example 2
Oxygen Consumption, Carbon Dioxide Production, and Respiratory Quotient Measurement (Metabolic Chamber Study)
The mice of the example 1 were fed with experimental diet for 5 weeks. The mice were then acclimatized in Oxymax System metabolic chambers (AccuScan Instruments, Inc. Columbus, Ohio USA) for 6 days with free access to the respective diet and water in an animal room kept on a 12 hour light and 12 hour dark cycle (light phase 8:00-20:00; dark phase 20:00-8:00) until steady. After acclimatization, data of the O2 consumption and CO2 production of the mice were automatically monitored and recorded for 24 hours on day 7 and the results were expressed as per gram body weight. The respiratory quotients (RQ) were also calculated as VCO2/VO2.
The results showed that the experimental group (the BGP group) at different time point of dark phase (20:00-8:00) showed significant increase of oxygen consumption (FIG. 1A) and CO2 production (FIG. 2A, p<0.05) per gram body weight. Further calculation of area under curve (abbreviated as AUC) of FIG. 1 and FIG. 2A indicated that the BGP group showed significant increase of oxygen consumption (FIG. 1B) and CO2 production (FIG. 2B, p<0.05) in the dark phase as compared to basal group. The VCO2 was further divided by VO2. The obtained results, respiratory quotient (abbreviated as RQ values), were shown in FIGS. 3A and 3B. The RQ values of the BGP group were 0.8-0.9, whereas the RQ values of the basal group were 0.7-0.8. Compared to the basal group, mice in the BGP group had significantly higher VO2 and VCO2 at quite a few time points in the dark phase (p<0.05). However, there is no significant difference (p>0.05) between the BGP group and the basal group in oxygen consumption, CO2 production, and RQ value when these groups were in the light phase (8:00-20:00).
Therefore, oxygen consumption, CO2 production, and RQ value of the BGP group through the dark phase was significantly higher than that of the basal group, indicating that respiratory activity of the BGP group mice was higher in the dark phase and energy and heat generation efficiency of mitochondria in the BGP group mice has been enhanced. This assumption requires further gene expression analysis to provide objective evidences.
Example 3
Gene Expression Analysis
At the end of 22 week of feeding, mice were feed-deprived at 3 AM in the morning for 16 hours, weigh of body weight and then sacrificed by CO2 asphyxiation. Excise and collect about 0.1 gram of epididymal white adipose tissue (abbreviated as EWAT), gastrocnemius muscle white adipose tissue and intra scapular brown adipose tissue in sterile 1.5 mL centrifuge tube or foil, then weighed and immediately frozen in liquid nitrogen, and then stored at −80° C. until gene expression analysis.
The mRNA expression of the following genes in epididymal white adipose tissue, brown adipose tissue (abbreviated as BAT) or muscle cells were analyzed, including PPARα (SEQ ID NO:1), PPARγ (SEQ ID NO:2), PPARδ (SEQ ID NO:3), and PGC1α (SEQ ID NO:4) genes; fatty acid oxidation related genes: CPT1a (SEQ ID NO:5), CPT1b (SEQ ID NO:6), ACD1 (SEQ ID NO:7), and ACS1 (SEQ ID NO:8); adaptive heat generation related gene: uncoupling protein 1 (UCP1 (SEQ ID NO:9)); cholesterol metabolism related gene and glycolysis related genes: SREBP1 (SEQ ID NO:10) and GK (SEQ ID NO:11); and mitochondria biosynthesis genes NrF1 (SEQ ID NO:12) and tfam (SEQ ID NO:13), wherein PCR primer and probes were purchased from Applied Biosystems.
RNA was isolated using TRIZOL reagent (Invitrogen) according to the instruction. Total RNA (2 μg) was reversed transcribed into cDNA in mixed solution containing 10×Rnt buffer, 100 mM dNTP mix buffer, 10×RT random primer, and MutiScribeTM RTase. Total volume of the polymerase chain reaction (PCR) mixture was 25 μL, containing 10 μL cDNA, 12.5 μL TaqMan® Gene Expression Master Mix, 1.25 μL probe/primer reagent mixture and water. PCR and fluorescence analysis was performed using ABI PRISM7000 Gene Sequence Detection System. Amplification conditions 150 were: 2 min at 50° C., 10 min at 95° C. then 40 cycles of 15 s at 95° C. and 1 min at 60° C.
Referring to FIG. 4A, in the epididymal white adipose tissue, the BGP group showed significant increase (p<0.05) in expression of PPARγ (SEQ ID NO:2), PGC1α (SEQ ID NO:4), fatty acid oxidation gene (CPT1a (SEQ ID NO:5), ACD1 (SEQ ID NO:7)), and uncoupling protein gene 1 UCP1 (SEQ ID NO:9), and Mitochondria biosynthesis gene (NrF1 (SEQ ID NO:12)). Referring to FIG. 4B, in the brown adipose tissue the BGP group showed significant increase (p<0.05) of expression in PPARδ (SEQ ID NO: 3), PGC1α (SEQ ID NO:4) and NrF1 (SEQ ID NO:12) genes. Referring to FIG. 4C, in the gastrocnemius muscle the BGP group also showed significant increase of expression (p<0.05) of PGC1α (SEQ ID NO: 4), ACS1 (SEQ ID NO:8) and tfam (SEQ ID NO:13) genes.
In summary of the examples above, the results demonstrate that male C57BL/6J mice fed with experimental diet for 22 weeks (the BGP group) show significant increase of oxygen consumption, CO2 production and AUCs. In the aspect of RQ value, the value of the BGP group is close to 1.0, compared to 0.7 of the Basal group. RQ value is the CO2 produced (mole)/O2 consumed (mole) when carbohydrates or fatty acids are metabolized. The RQ value of glucose oxidation is about 1 and RQ value of fatty acid is around 0.7. Therefore, the BGP group shows significant increase of carbohydrate oxidation when in the dieting phase (dark phase).
Experiments are designed to determine gene expression in gastrocnemius muscle, brown adipose tissue and epidiymal white adipose tissue. The results indicate that BGP group shows significant increase expression of PGC1α (SEQ ID NO: 4), fatty acid metabolism related gene CPT1a (SEQ ID NO: 5) and ACD1 (SEQ ID NO:7), mitochondria biosynthesis gene NrF1 (SEQ ID NO:12) and UCP1 (SEQ ID NO:9) in epididymal white adipose tissue. On the other hand, BGP shows significant increase of expression of PGC1α in gastrocnemius muscle and brown adipose tissue as compared to the control group.
Because PGC1α (SEQ ID NO:4) can regulate heat generation in brown adipose tissue, mice lack of PGC1α (SEQ ID NO:4) expression will die when exposed at low temperature environment due to low body temperature. On the other hand, increase of PGC1α (SEQ ID NO:4) expression in white adipose tissue can enhance activity of mitochondria and increase expression of UCP1 (SEQ ID NO:9). Therefore, the results demonstrate that BGP group mice have enhanced PGC1α (SEQ ID NO:4) gene expression in muscle cell, enhanced PGC1α (SEQ ID NO:4), NrF1 (SEQ ID NO:12) or tfam (SEQ ID NO:13) gene expression in brown adipose tissue cell, and increased PGC1α (SEQ ID NO:4), NrF1 (SEQ ID NO:12) or tfam (SEQ ID NO:13) in white adipose tissue cell, such that mitochondria biosynthesis in the above tissues also increased. Because of this, the test results of the present invention BGP group mice show higher oxygen consumption in the ingestion phase (dark cycle), CO2 production, and energy expenditure, i.e., an increase in RQ value.
Therefore, the mitochondria regulator composition of the present invention can be used as a regulator of mitochondria biosynthesis, or used as an alternative or supplement for control of heat and energy generation. For example, the mitochondria regulator composition of the present invention can be administered an effective amount in treatment of symptoms caused by thermogenin deficiency, the number of mitochondria deficiency, mitochondrial dysfunction, mitochondria malfunction or mitochondria dysregulation; and the mitochondria regulator composition further comprises a pharmaceutical acceptable carrier. In addition, the mitochondria regulator composition of the present invention can be used as a food composition for improvement symptoms of thermogenin deficiency, the number of mitochondria deficiency, mitochondrial dysfunction, mitochondria malfunction or mitochondria dysregulation. The food composition can further contain an additive, wherein the additive can be but not limited to a healthy ingredient, a food ingredient or the combination thereof. The above healthy ingredient can be citric acid, taurine, vitamins, pantothenate, niacin or any other ingredients that may benefit health. The food may be selected from, but not limited to, vegetables, fruits or meats.

## Claims

1. A composition for the regulation of mitochondria biosynthesis consisting essentially of therapeutically effective amounts of whole bitter gourd fruit extract, corn starch and cellulose.