CN111134295A

CN111134295A - Preparation of high content synephrine extracts from immature citrus fruits, method and use thereof

Info

Publication number: CN111134295A
Application number: CN201811465867.9A
Authority: CN
Inventors: 林丽云; 庄正宏; 黄柏彰
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-11-05
Filing date: 2018-12-03
Publication date: 2020-05-12
Also published as: TWI713924B; TW202017582A

Abstract

The invention relates to an extract containing high-content synephrine which is separated and purified from immature citrus fruits and a preparation method thereof. The high-content synephrine extract prepared by the invention can replace ephedrine and is applied to preparing supplements for weight control and blood fat reducing health care products by mixing with other compound materials.

Description

Preparation of high content synephrine extracts from immature citrus fruits, method and use thereof

Technical Field

The invention relates to a method for extracting synephrine from citrus fruits. More particularly, the present invention relates to a method for separating and purifying high-content synephrine extracts from unripe citrus fruits or pericarps.

Background

In recent years, the living standard of people in China is improved, diet is influenced by western countries, the dietary habits are gradually changed into high oil, high sugar, high calorie and refined forms, however, the food indirectly causes overhigh concentration of Low Density Lipoprotein (LDL) or overlow concentration of High Density Lipoprotein (HDL) in blood, causes atherosclerosis and insulin resistance, easily causes blood sugar and blood lipid abnormality to increase oxidation pressure, has close correlation with peroxidation of free radicals and fat, and causes the increase of disease suffering rates of vascular diseases, second diabetes and the like, according to the statistics of health and welfare department of 105 years, the heart diseases, the cerebrovascular diseases and the diabetes of ten causes of death of people in China respectively occupy the second place, the fourth place and the fifth place, and are caused by interaction of multiple factors, but obesity is the most main cause of the diseases. Therefore, under the growing health consciousness and the popularization of preventive medicine concept, the utilization of natural antioxidants and medicinal plants is increasingly gaining attention.

In the supplement field, 75% of products are supplements formed by mixing synephrine and other stimulants (such as caffeine, green tea and cocoa). the synephrine has the effects of boosting pressure, resisting shock, improving metabolism and improving basic metabolism, is most representative of weight reduction in recent research and can promote sympathetic nerve activity, increase thermogenesis and stimulate lipolysis to achieve fat oxidation, and the mechanism is presumed that the synephrine can act on β 1-, β 2-, β 3-and α -adrenoreceptors to rapidly oxidize fat.

Citrus fruits are one of the international large fruit crops, and the annual yield in taiwan is about 47 ten thousand tons, and the pericarp, fallen fruit or thinned fruit of citrus fruits contain many active substances, such as synephrine, essential oil, phytosterol, limonin, phenolic acid, flavonoid and the like. In recent years, the chemical structure of synephrine has been proved to be similar to that of ephedrine, both of which have the effect of reducing weight, however, ephedrine has limited weight-reducing effect and has excessive side effects (such as causing anxiety, hypertension and the like), and is prohibited from being used in dietary supplements by the U.S. FDA in 2004.

It is known that the fruit of lime (Citrus aurantium l., also known as bitter orange) contains the major active ingredient synephrine, and that extracts thereof have been widely used for appetite suppression and dietary supplements, while lime fruit is used in various juices and foods. Many studies have shown that lime fruit extracts are safe health Food additives (M.D. arbor et al, Food and pharmaceutical nomenclature.46, 2770-2775,2008; Marcelo Dutra arbor et al, Regulatory nomenclature and pharmacy.54, 114-117,2009; N.S. Deshmukh et al, nomenclature reports.4,598-613,2017).

The pericarp, fruit drop and fruit thinning of citrus fruit are usually discarded as waste or disposed of as fertilizer, with few other uses. The citrus fruits have two situations of physiological fruit drop in the young fruit period, and the first situation is that pollination is incomplete, so that embryonic plants are developed or malnutrition is caused; the second is caused by endogenous hormones in the early-maturing embryo strains. In addition, if the amount of fruit bearing is too large, farmers may reduce the excessive load on the plants by thinning and appropriately picking off the late-flowering fruits. However, the number of immature fruits in fruit drop and thinning is quite large, and only a small amount of immature fruits are used in traditional medicine, and most of immature fruits are burned as fertilizer or agricultural waste.

Disclosure of Invention

In view of the above, the main object of the present invention is to provide a method for preparing synephrine extract with high content from unripe fruit part of citrus fruit.

Accordingly, one aspect of the present invention is directed to a method for preparing a synephrine-rich extract from unripe citrus fruit parts, said method comprising:

taking dried immature citrus fruit or pericarp thereof;

extracting with ultrasonic wave, stirring or microwave method and methanol as solvent, wherein the ratio of the weight of dried fruit or pericarp to the volume of methanol is 1:8-1:10 g/ml;

repeating the extraction step for 2 times for the residue after centrifugation and filtration;

combining the extract liquor; and

the combined extracts are concentrated to remove the solvent to obtain an synephrine-rich extract.

In some embodiments of the invention, the immature citrus fruit comprises young fruit, fallen fruit and thinning fruit.

In one embodiment of the invention, the immature citrus fruit part is the exocarp of a citrus fruit in the young fruit stage.

In another embodiment of the present invention, the dried unripe citrus fruit or pericarp is ground prior to extraction.

In another embodiment of the invention, the methanol is added in an amount of 5-10 ml/g dry weight of the fruit.

In other embodiments of the present invention, the ultrasonic method is performed by ultrasonic oscillation at 4-25 ℃ for 20 minutes-2 hours.

In other embodiments of the invention, the stirring is carried out at an extraction temperature of 25-28 ℃ for 20 minutes to 2 hours.

In other embodiments of the present invention, the microwave process is an extraction in a closed microwave-assisted extraction system at an elevated temperature of 30 ℃ to 64 ℃ for 10-15 minutes.

In other embodiments of the invention, the ratio of the weight of the dried fruit or peel to the volume of methanol is 1:8 g/ml.

Another aspect of the invention relates to a high content synephrine extract prepared according to the method of the invention.

In some embodiments of the invention, the high content of synephrine extract is from the exocarp of citrus fruit during the young fruit stage.

In some embodiments of the invention, the immature citrus fruit part is the epicarp of the young fruit.

In another aspect, the present invention relates to a composition for weight management comprising a high content of synephrine extract prepared according to the method of the present invention.

In another aspect, the present invention relates to a composition for reducing blood lipid, comprising the high content of synephrine extract prepared by the method of the present invention.

The method for preparing the high-content synephrine extract from the immature citrus fruit part effectively extracts the synephrine in the immature citrus fruit by using the immature citrus fruit peel, the fruit drop and the fruit thinning fruit as raw materials, and further uses the high-content synephrine extract for researching and developing health-care food or compound food, so that the purpose of reducing environmental pollution caused by wastes can be achieved, and the economic value of the immature citrus fruit peel, the fruit drop and the fruit thinning fruit can be increased. In addition, the content of synephrine in citrus fruit varies with the ripening months and is inversely proportional to the ripeness of the citrus fruit and the fruit diameter.

Drawings

Fig. 1 shows the yield of seedless lemon pericarp extracted by ultrasonication and various solvents (methanol (100% (v/v)), ethanol (95% (v/v)) and water), the yield (%) being the extract weight after concentration/pericarp weight 100%.

Fig. 2 shows the yields obtained by extracting seedless lemon peels with stirring and with different solvents (methanol (100% (v/v)), ethanol (95% (v/v)) and water), yield (%) 100% of the concentrated extract weight/peel weight.

Fig. 3 shows the yields obtained by extracting seedless lemon peels using a microwave method and various solvents (methanol (100% (v/v)), ethanol (95% (v/v)) and water), the yield (%) being the extract weight after concentration/peel weight 100%.

FIG. 4 is an HPLC chromatogram of a synephrine standard and a shaddock peel concentrated extract, wherein a represents the synephrine standard, and b and c are HPLC chromatograms of shaddock peel concentrated extracts of different batches.

FIG. 5 shows the content of synephrine in concentrated extracts obtained from seedless lemon pericarp at the young fruit stage, the strong fruit stage and the mature stage using ultrasonic method, stirring method, microwave method and methanol solvent.

Detailed Description

For a better understanding and appreciation of the objects, features, and advantages of the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings.

The "citrus fruit" referred to in the specification includes species such as orange, tangerine, grapefruit, kumquat, lemon, and the like.

The fruit of the plant has a protective function for the seeds and can help the seeds to spread after the seeds are mature. The exocarp of citrus fruit consists of an outer epidermal layer, oil glands and parenchyma cells containing crystals; the mesocarp contains a large number of parenchyma cells, vascular tissues among the parenchyma cells are in a net distribution, and the tangerine pith is obtained after the fruit is mature. Under normal climatic conditions, the ripening process and progress of the fruit can be indicated by changes in the color of the peel. For example, the late maturing varieties in most sweet oranges, wide-skinned oranges, turn green and yellow to 2/3, which is considered to have reached eighty percent maturity. As used herein, the term "immature citrus fruit" refers to fruit that has not reached maturity criteria, preferably citrus fruit at the young fruit stage.

EXAMPLE one, different extraction methods and solvents for preparing high content sinephrine extracts

In this embodiment, the seedless lemon pericarp is extracted by using ultrasonic waves, stirring, microwaves and different solvents (methanol, ethanol and water) to obtain an extract, and the obtained yield (percent) is calculated as the weight of the concentrated extract per pericarp and 100 percent), so that how much extract can be extracted from 100g of pericarp can be known, and the content of synephrine in the extract can be further analyzed and converted to establish the condition for extracting the synephrine by using the optimal solvent.

An ultrasonic extraction method (Shiming Li et al, 2006, Journal of agricultural and food chemistry.54(12),4176-4185) was performed by adding 8mL of methanol (100% (v/v)), ethanol (95% (v/v)) and an aqueous solution to 1g of dried seedless lemon powder, oscillating the mixture for 20 minutes to 2 hours (extraction temperature: 28 ℃) with ultrasonic waves, centrifuging the mixture, adding the residue to the solvent for extraction, filtering the mixture with NO.1 filter paper, combining the two filtrates in a concentration bottle, concentrating the mixture at 40 ℃ under reduced pressure, quantifying the mixture to 10mL, and filtering the mixture with 0.22 μm filter membrane to 1 mL.

The stirring extraction method comprises adding 1g dried Seedless lemon powder into 8mL methanol (100%), ethanol (95%) and water solution respectively, stirring with a stirrer for 20 min-2 hr (extraction temperature: 28 deg.C), centrifuging, adding the above solvent into the residue, extracting, filtering with NO.1 filter paper, mixing the two filtrates, concentrating at 40 deg.C under reduced pressure to give 10mL, filtering with 0.22 μm filter membrane to give 1 mL.

Referring to Inoue et al, 2010, Food Chemistry,123: 542-; the ethanol solvent condition was 30 ℃ raised to 78 ℃ (15 minutes) and maintained for 30 minutes; raising the temperature of the methanol solvent to 64 ℃ (12 minutes) under the condition of 30 ℃, maintaining the temperature for 30 minutes, centrifuging, adding the residue into the solvent for extraction, filtering by using NO.1 filter paper, combining the two filtrates into a concentration bottle, controlling the temperature to be 40 ℃, carrying out reduced pressure concentration, quantifying to 10mL, and filtering 1mL by using a 0.22 mu m filter membrane. As shown in fig. 1, 2 and 3, the extraction using methanol as the solvent can achieve significantly higher extraction rates in the ultrasonic method, the stirring method and the microwave method than those using ethanol and water as the extraction solvent.

Therefore, as a preferred embodiment of the present invention, immature citrus fruits are dried (drying conditions: drying with a 50 ℃ hot air dryer to a moisture content of 10% or less) and then subjected to ultrasonic, agitation or microwave extraction with methanol (the ratio of the weight of the fruits to the volume of methanol is about 1:8 g/ml). The extraction mixture was then centrifuged at 3000rpm for 15 minutes and filtered. The methanol ultrasonic extraction step was repeated 2 more times on the obtained residue. The extracts obtained in the respective steps are combined and concentrated (concentration condition: concentration is carried out by a concentration drier at 60 ℃ until no solvent exists), and the solvent is removed to obtain a concentrated extract. (see Li et al (2006) for extraction of synephrine, phenolic compounds, and flavonoids).

Example II analysis of the extracts for the content of synephrine

In this example, the content of synephrine in the concentrated extract was measured by HPLC analysis. Firstly, the synephrine standard substance with different concentrations is analyzed by HPLC, and a standard curve is drawn according to the concentration and the area so as to calculate the content of the synephrine in the sample. The conditions of the HPLC apparatus and the chromatographic column for analysis were as follows: the instrument specification is as follows: HITACHI Pump L-2130; a detector: HITACHI UV Detector L-2400; analysis software: HITACHI Model D-2000 EliteChromograph Data Station Software; separating the tubular column: RP-18GP250 (250X 4.6mm, Mightysil, Kanto Chem Co., Inc.); mobile phase: a.10% (v/v) methanol solution with addition of 0.05% (v/v) formic acid, B.100% (v/v) methanol solution with addition of 0.05% (v/v) formic acid; setting the flow rate: 1.0 mL/min; sample injection amount: 10 mu L of the solution; UV light wavelength: 280 nm; mobile phase gradient:

time (min)	A(％)	B(％)
			0	100	0
5	80	20
			35	45	55
70	35	65
			75	35	65
80	0	100
			85	0	100
90	100	0
			95	100	0

The HPLC analysis of the synephrine standard and the peel of pomelo is shown in FIG. 4. The results show that the comparison of the synephrine standard and the peel of the pomelo proves that the peel of the pomelo also contains the synephrine compound, and the time position of the synephrine in the peel of the pomelo can be confirmed so as to facilitate the subsequent collection and purification of the solution of the synephrine.

Example III analysis of the content of Indenofurin in Citrus fruits at young fruit stage, strong fruit stage and mature stage

Concentrated extracts were prepared from pomelo, orange, ponkan and seedless lemon fruits at the young, strong and mature fruit stages, respectively, by the methanol ultrasonic extraction method described in example one, and analyzed and calculated for the content of synephrine by HPLC. See table 1 below for results.

TABLE 1 variation of the content of sinephrine in young fruit, strong fruit and mature fruit of four citrus fruits

¹The values in the table are all expressed as mean. + -. SD, significant differences (p) between the values<0.05, n ═ 3) series were analyzed using Duncan's range test

²The unit is fresh citrus fruit weight

³The unit is mg/100 g dry weight of pericarp

As shown in Table 1, the content of synephrine in pomelo, orange, ponkan and seedless lemon in the young fruit stage is significantly higher than that in the strong fruit stage and the mature stage. The cells of the tissues of various parts inside the young fruit are undergoing vigorous cell division, wherein the pericarp thickening speed is the fastest. In the fruits in the strong fruit period and the mature period, the juice cells grow and are shaped, the juice is rapidly increased, the weight of the fruits is obviously increased, the color of the fruits begins to change due to the decomposition of chlorophyll, the accumulation of carotenoid, anthocyanin and exocarpium citri rubrum, and the aroma is also improved along with the increase of the maturity. Therefore, the content of synephrine in citrus fruits varies according to the ripening months, and shows that the content is inversely proportional to the fruit diameter, so that if a higher content of synephrine extract is desired, it is necessary to use young fruits with low ripening as raw materials.

Example four Effect of different extraction methods on the content of Indomalin in Seedless lemon pericarp

Using the ultrasonic, stirring and microwave extraction method described in example one and methanol as an extraction solvent, concentrated extracts were prepared from seedless lemon pericarp in the young fruit stage, the strong fruit stage and the mature stage, respectively, and the content of synephrine in the extract was analyzed and calculated by using HPLC described in example two. The results are shown in FIG. 5, which shows that the concentration of sinephrine in the concentrated extract obtained by stirring extraction is higher than that obtained by microwave extraction and ultrasonic extraction.

Example five evaluation of weight control and hypolipidemic efficacy of high content synephrine extracts

This example was conducted to test the effect on weight gain and hyperlipidemia induced by high fat diet in rats by obtaining synephrine-rich extracts and essential oils from the peel of pomelo, seedless lemon during the young fruit stage according to the procedure described in example one.

Source, feeding, dosing and sampling of experimental animals (high-fat diet mice)

Male Wistar rats of 6 weeks of age of leszidae biosciences were used as test animals for weight, blood lipid, and body lipid regulation, and 76 Wistar rats were purchased. The weights were grouped on the first day of purchase, assigned to mean standard deviation of each group (p <0.05), and after 1 week of acclimation, they were grouped according to weight at week 2, with 8 groups in total, except for 12 control groups, and 9 groups. Rats were housed in stainless steel cages with the animal house temperature set at 22 + -2 deg.C and relative humidity at 40-60%, with automatic timer control of light time, light time (light period) 8:00 in the morning to 8:00 in the evening, dark time (dark period) 8:00 in the evening to 8:00 in the morning, and feed and drinking water were taken from the house. During the experiment, food intake and water intake were recorded daily, body weight was measured 2 times per week, blood was collected in the eye socket 1 time per 2 weeks, plasma was centrifuged at 4 ℃ (1500rpm) for 20min, serum was taken out to determine changes in TG, TC, HDL and LDL, and sacrificed after 12 weeks of rearing, while feces were collected for 3 days in each group on average before sacrifice, rats were fasted for 12h before sacrifice, and coma and sacrifice were performed with isoflurane. Before sacrifice, the blood of the eye fossa is collected and weighed, and then the blood of the abdominal aorta, visceral organs (including kidney and liver) and visceral adipose tissues (including perirenal fat and accessory testis fat) are collected during sacrifice. Blood is centrifuged for 20min at 4 ℃ (1500rpm) (KUBOTA-374), serum is collected, the serum in the orbital part is used for detecting the content of TG, TC, HDL and LDL, the serum in the abdominal aorta is sent to Dahua medical examination center in Zhanghuaxian county of China for detecting insulin (insulin) and thin voxel (leptin), after the liver and visceral fat of each group are cleaned by PBS, part of the liver is rapidly frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for the content measurement of TG and TC in the liver, and the rest is put into 10% formalin solution for subsequent examination.

Preparing high-fat diet feed:

the Control group (C: Control group) was fed with AIN-76 formula (AIN,1977) feed; the High fat High cholesterol diet (HF: High fat) was fed with a feed based on the AIN-76 formula plus 10 wt% lard, 5 wt% corn oil and 0.2 wt% cholesterol; positive control (Positive control) was fed a feed based on AIN-76 formulation plus an additional 10% lard, 5% corn oil, 0.2% cholesterol and p-synephrine (15mg/kg/B.W.) dosage referenced to arbor et al (M.D. arbor et al, Food and Chemical society.46, 2770-2775,2008); the experimental group of pomelo is based on AIN-76 formula, 10 wt% of lard oil, 5 wt% of corn oil and 0.2 wt% of cholesterol are added, then pomelo non-essential oil peel powder and essential oil are added respectively, the experimental group (excluding positive control group) is divided into low dose and high dose groups, and the prepared feed is stored in a refrigerated cabinet at 4-6 ℃ for standby after 12 weeks.

Determination of triglycerides:

this experiment determined that triglycerides in serum were analyzed with Randox kit (cat. No. tr213). And (3) putting 2 mu L of each group of serum samples into a 96-hole plate, adding 200 mu L of triglyceride buffer in the dark, uniformly mixing, standing for 10min at room temperature, measuring the light absorption value of the sample with the wavelength of 500nm by using an ELASA reader, and converting the concentration of the triglyceride in the sample by referring to a product providing formula. The reference formula is as follows:

triglyceride (mg/dL) ═ A (sample abs/A standard abs). Standard conc (148.6)

Determination of cholesterol:

the total cholesterol in serum determined in this experiment was analyzed using Randox kit (cat. No. ch201). And (3) putting 2 mu L of each group of serum samples into a 96-hole plate, adding 200 mu L of cholesterol reagent in the dark, uniformly mixing, standing for 10min at room temperature, measuring the light absorption value of the sample with the wavelength of 500nm by using an ELASA reader, and converting the concentration of the cholesterol in the sample by referring to a product providing formula. The reference formula is as follows:

cholesterol (mg/dL) ═ A sample abs/A standard abs standard conc (110.6)

Determination of low density lipoprotein:

this experiment determines low density lipoprotein cholesterol in serum which is analyzed using Randox kit (Cat. No. KGLCH103). Taking 50 mu L of each group of serum samples, adding 500 mu L of LDL precipitator, uniformly mixing, standing for 10min at room temperature, centrifuging by a low-temperature centrifuge (1500 rpm; 15min), taking 12.5 mu L of supernatant, adding 250 mu L of cholesterol reagent, uniformly mixing, standing for 10min at room temperature, measuring the light absorption value of the sample with the wavelength of 500nm by using an ELASA reader, and converting the concentration of the low-density lipoprotein cholesterol of the sample by referring to a product providing formula. The reference formula is as follows:

LDL (mg/dL) ═ (A sample abs/A standard abs). Standard conc (316.25)

Determination of insulin and leptin:

the Dahua medical examination institute in Zhanghua county was entrusted with inspection.

Animal formula: animal feed formulations refer to Feng et al (Li-Jun Feng et al, Food research International 44,404-409,2011 for details) and AIN-76 formulations (see Lien EL1, et al, Food Chem Toxicol 39(4):385- "392, 2001). The following animal formula tables are provided, and the synephrine in the peel extract of the pomelo and the seedless lemon is used as the drug administration target, and the non-toxic dose of the synephrine is recorded in the literature, and the addition of the peel extract and the essential oil of the two citrus fruits is converted into the required content of the high-fat diet formula. The detailed formulation is shown in tables 2 and 3.

In table 3 below, C: a control group given a normal diet; HF: a group of high fat diets given a high oil and high cholesterol diet; synephrine: the treatment group was given a high oil and high cholesterol diet containing 0.015% synephrine; GPF-L: the treatment group was given a high oil and high cholesterol diet containing 6.8% of grapefruit peel extract; GPF-H: the treatment group was given a high oil and high cholesterol diet containing 20.4% of grapefruit peel extract; GPEO-L: administering a low dose treatment group containing a high oil and high cholesterol diet containing 0.04% grapefruit essential oil; GPEO-H: the high dose treatment group was given a high oil and high cholesterol diet containing 0.16% grapefruit essential oil.

TABLE 3 Wen Dan shaddock-animal feed formulation (unit: weight percent)

In table 4 below, C: a control group given a normal diet; HF: a group of high fat diets given a high oil and high cholesterol diet; synephrine: a treatment group given a high oil and high cholesterol diet containing 0.00625 wt% synephrine; LEON: the treatment group was given a high oil and high cholesterol diet containing 0.744 wt% of seedless lemon essential oil; LEOH: the treatment group was given a high oil and high cholesterol diet containing 2.232 wt% seedless lemon essential oil; LPN: the treatment group was given a high oil and high cholesterol diet containing 24.2 wt% of seedless lemon peel extract; LPH: the treatment group was given a high oil and high cholesterol diet containing 72.6 wt% of seedless lemon peel extract.

TABLE 4 Seedless lemon-animal feed formulation (unit: weight percent)

As a result:

A. weight effect of high content of synephrine extract (Peels) and Essential Oil (EO) on high fat diet rats of pericarp of Pomelo (Pomelo) and seedless lemon (Lime)

The results show that the initial average body weight of each group of rats fed with pomelo and lemon diets has no statistical difference after statistical analysis, and after 12 weeks of high-fat high-cholesterol experiment, the final body weight of each group measured before sacrifice can find that the average body weight of the high-fat group of the pomelo and lemon diet experiment is increased by 440.8 +/-0.08 g and 361.3 +/-5.33 g respectively, which are obviously higher than that of the control group, the positive control group, the pericarp group and the essential oil group. Wherein the weight of the Xinnevirin group of the pomelo diet is reduced by 93.6g compared with the high-fat group; the weight of the low-dose and high-dose pericarp groups was reduced by 114.8g and 113.9g, respectively; the body weight of the low dose and the high dose of the essential oil group was reduced by 93g and 119.2g, respectively. Compared with the high-fat group, the weight of the synephrine group with the lemon diet is reduced by 32 g; the weight of the low dose and high dose peel groups was reduced by 305.8g and 467.5g, respectively; the body weight of the low dose and the high dose of the essential oil group was reduced by 71.9g and 171.9g, respectively.

The main compound of the essential oil, namely the Limonene, can regulate the expression of uncoupling protein-2 (UCP-2) and sterol-regulatory element binding protein (SREBP-1), wherein the uncoupling protein-2 and the sterol-regulatory element binding protein (SREBP-1) are important factors for fatty acid oxidation and triglyceride synthesis respectively, and the Limonene (Limonene) induces the essential oil to improve fat metabolism. In addition, limonene can improve the circulatory system, discharge carbon dioxide and water, and allow the lymphatic system to circulate and normally discharge waste. The synephrine, dietary fiber and flavonoid in the peel have relevant influence on weight control, wherein the synephrine can stimulate adrenaline to influence the action of cardiovascular system, the action can generate heat in human body to help reduce body fat, and the active ingredients in the citrus peel and essential oil thereof can effectively reduce the increase of weight.

B. Effect of high content of synephrine extracts (Peels) and Essential Oil (EO) on Triglyceride (TG) content in high fat diet rat of pericarp of Pomelo (Pomelo) and seedless lemon (Lime)

The results show that the initial mean triglyceride of each group of rats fed with pomelo and lemon diets was statistically different after statistical analysis, while the triglyceride content of the serum of the high-fat group was 173.73mg/dL and 114.53mg/dL in the high-fat group at weeks 6 and 4 respectively in the high-fat diets of pomelo and lemon, which indicates that the induction of the triglyceride content in the high-fat group was successful. After a 12-week feeding of pomelo and lemon diets, the high dose group of essential oils was found to be the best, probably the most essential compound limonene, which prevented the development of dyslipidemia in mice (sting et al, 2013). The pomelo and lemon peel groups are optimized at low dose, the possible reason for effectively reducing serum triglyceride is the nomilin compound, the feeding of 0.2% (w/w) nomilin can find that the body weight, serum glucose and insulin of mice with high fat diet are obviously reduced and the glucose tolerance is improved, and the active ingredients in the citrus peel and essential oil thereof can effectively reduce the increase of serum triglyceride.

C. Effect of high content of synephrine extracts (Peels) and Essential Oil (EO) on total cholesterol levels in high fat diet rats by pericarp of Pomelo (Pomelo) and seedless lemon (Lime)

The data in the above table show that the initial average total cholesterol of all groups of rats fed with pomelo and lemon diets was not statistically different after statistical analysis, while the triglyceride content in the serum of the high-fat group was 75.53mg/dL and 69.45mg/dL highest at weeks 6 and 4 respectively in the high-fat diets of pomelo and lemon, indicating that the total cholesterol content in the high-fat group was successfully induced in the experiment. After 12 weeks of feeding the diets of pomelo and lemon, the synephrine group, pericarp group and essential oil group were found to be effective in inhibiting cholesterol production. It has been suggested in the literature that an increase in the number of low density lipoprotein receptors resulting from inhibition of HMG-CoA reductase and ACAT activity is effective in inhibiting cholesterol production and reducing serum low density lipoprotein concentration (Nicolosi et al, 1997). In conclusion, the active ingredients in the citrus peel and essential oil thereof can effectively reduce the increase of total cholesterol in serum.

D. Effect of high content of synephrine extracts (Peels) and Essential Oil (EO) on the low density lipoprotein content of rats with high fat diet of peel of Pomelo (Pomelo) and seedless lemon (Lime)

The data in the table above shows that there was no statistical difference in the initial mean low density lipoprotein for each group when rats were given a grapefruit and lemon diet. The low-density lipoproteins contained a large amount of cholesterol, and the low-density lipoproteins in the serum of the high-fat diet groups of the pomelo and lemon experiments were 34.14mg/dL and 39.2mg/dL at week 6 and 4, respectively, which were highest, so that the low-density lipoproteins in the high-fat group of the experiment were higher than those in the other groups. The results show that both the pericarp group and the essential oil group are effective in reducing the low density lipoprotein content. The literature indicates that the citrus flavonoid and the compound with strong oxidation resistance form the formula food, can effectively reduce the content of 40 percent of low-density lipoprotein, and simultaneously inhibit cholesterol from forming fatty liver in the liver. Wherein the hypermethoxyflavonoids in the pericarp can reduce the serum very low density lipoprotein and low density lipoprotein of high fat diet hamster (Kurowska et al, 2004; Liet al, 2006).

E. Effect of high content of synephrine extract (Peels) and Essential Oil (EO) on triglyceride and total cholesterol content in liver of high fat diet rat

It was found that administration of a diet containing more than 0% cholesterol resulted in a positive correlation between liver and plasma cholesterol levels and the levels of additive, and a reduction in β -oxidation-related creatine transferase I activity, and a retardation in fatty acid metabolism, leading to a sustained triglyceride production, which was delivered to the liver from very low-density lipoproteins, whereas the very low-density lipoproteins were not increased, resulting in a significant accumulation of triglycerides in the liver to form fatty liver (Fungwe et al, 1992; Liuet al, 1995). the data in the above table show that the total cholesterol and triglyceride levels in the liver of the high-lipid group of the grapefruit and lemon diet experiment were as high as 70mmol/L or higher, which is significantly different from those in the normal diet control group of 48.89-58.36, 46.9556.95mmol/L (p <0.05), which tends to cause the accumulation of fat in the hepatocytes, leading to fatty liver development, and that the total cholesterol level in the high-lipid group of grapefruit and the low-lipid group of the grapefruit diet was found to be lower than those in the high-cholesterol group, the low-cholesterol level of 48.59, 35% and 35.35% of the low-lipid group, and the low-lipid group was found to be lower than that the diet group of the low-lipid group, respectively, and the low-lipid group was found to be lower than that the diet.

When triglyceride is compared with high oil and fat in experimental group of shaddock, the low dose and high dose of pericarp group are respectively reduced by 6.58% and 29.18%; the low dose and the high dose of the essential oil are respectively reduced by 19.19 percent and 26.63 percent. When triglyceride is compared with high oil and fat in the experiment group of lemon diet, the low dose and the high dose of the pericarp group are respectively reduced by 36.26 percent and 40.84 percent; the low dose and the high dose of the essential oil group are respectively reduced by 23.14 percent and 27.64 percent. Among them, the two citrus fruits are most remarkable in reducing total cholesterol and triglyceride in liver at high dose of pericarp group, probably due to naringin, quercetin, hesperetin or polyphenol compound, and it is indicated in literature that hesperetin limits the increase of triglyceride and cholesterol in liver by the induction of orotic acid (Cha et al, 2001) or citrus chazukii polyphenol can activate cAMP-regulated kinase (AMKP) to activate liposome autophagy (lipoliver), preventing non-alcoholic fatty liver.

F. Effect of high content of synephrine extracts (Peels) and Essential Oil (EO) on fecal sterol content in high fat diet rats by pericarp of Pomelo (Pomelo) and seedless lemon (Lime)

Cholesterol is obtained by two routes, one is synthesized by the human body and the other is obtained from the diet, and the excretion mode also comprises two modes, namely cholesterol 7 α -hydrolase in the liver is excreted out of the body in the cholic acid mode after a series of reactions, and the excretion mode comprises cholesterol or plant sterol which is not absorbed or is excreted with bile, through stimulating cholesterol 7 α -hydrolase to increase acid cholesterol and neutral sterol in the excrement (Quintao et al, 1987). the data in the above table shows that the neutral sterol content in the excrement is 7.15mg/g in the high lipid group of the shaddock diet experiment, while the low and high dosages of the pericarp group are 8.38 and 14.06mg/g respectively, the low and high dosages of the essential oil group are 8.87 and 10.98mg/g respectively, the high lipid group of the lemon diet experiment is 7.56mg/g, while the low and high dosages of the pericarp are 7.61 and 11.56mg/g respectively, the low dosages of the essential oil group are 8.84 mg/g respectively, the lipid group of the lemon diet experiment is 7.56mg/g, the lipid group can prevent the bile acid from being absorbed in the intestinal tract, and the bile fiber can be absorbed by the intestinal tract, and the intestinal tract can be prevented from being accumulated in the intestinal tract, and the bile fiber can be effectively absorbed by the cholesterol absorption of the cholesterol.

G. Effect of high content of synephrine extracts (Peels) and Essential Oil (EO) on insulin and leptin content in high fat diet rats

Leptin is 167 amino acid peptides secreted by white adipose tissue, which modulates leptin effects via leptin receptors in the hypothalamus, while hyperphagia increases leptin content and conversely decreases leptin concentration, resulting in a concentration proportional to body fat. When leptin levels are reduced in vivo, central nerves and surrounding tissues are stimulated to signal increased appetite and reduced energy expenditure, conversely, when leptin levels are high, appetite is reduced. The results in the table show that the high fat group without pomelo and lemon diets contained 4.14 ng/mL and 8.28ng/mL, respectively, were the highest, and were significantly different from the pericarp and essential oil groups (p <0.05), indicating that high fat is likely to cause leptin resistance and fail to send signals to the central nervous system to increase energy consumption. The pericarp group and the essential oil group can effectively reduce the weight gain, so the lean body mass is lower than that of the high fat group.

Insulin is a hormone secreted by β cells of the pancreas, and stimulates organs in the body to store excess glucose and inhibit lipolysis of adipose tissue, but lipid accumulation in the abdomen is considered to be the main cause of systemic insulin resistance if it causes specific disappearance of insulin signaling pathway and impaired insulin regulation of glucose (Carey et al, 1996). the results listed in the above table indicate that 1.08 and 2.78ng/mL of high lipid group, which may have insulin resistance, are likely to have too high triglyceride and low density lipoprotein content in serum of high lipid group, and likely to have too much insulin secretion, resulting in abnormal blood fat metabolism (Chen et al, 2006). compared to the high lipid group, the pericarp group and essential oil group are significantly decreased (p <0.05), because insulin, in addition to metabolizing glucose, also affects fat metabolism, increases fat synthesis by liver and liver cells, decreases fat and triglyceride decomposition in muscle, and the experimental results indicate that the content of triglyceride in pericarp and essential oil group is lower than that normal glucose tolerance of insulin is achieved (Leey et al).

H. Effect of high content of synephrine extract (Peels) and Essential Oil (EO) on body fat content of rats with high fat diet from pericarp of Pomelo (Pomelo) and lemon (Lime)

Body fat includes essential fat and storage fat, while visceral fat is a storage fat that protects the viscera from damage, but is also prone to chronic disease. The experimental data in the table above show that in the high-fat group of the pomelo and lemon diet experiments, the visceral and testicular fat contents are 34.38g and 22.81g respectively; 32.52g and 20.44g were the highest because accumulation of adipose tissue correlated with the amount of triglyceride, which accumulated in visceral fat cells, increased the size of the cells and developed visceral fat obesity. Compared with the high-fat group, the low dose and the high dose of the peel group of the pomelo are respectively reduced by 9.67 percent and 19.18 percent, and the low dose and the high dose of the essential oil group are respectively reduced by 8.88 percent and 20.48 percent; the low dose and the high dose of the lemon peel group are respectively reduced by 73.27 and 100 percent, and the low dose and the high dose of the essential oil group are respectively reduced by 33.87 and 53.88 percent.

In summary, compared with untreated rats with high-fat diet, the immature citrus fruit peel extract of the present invention can effectively control the body weight and reduce the blood fat content of rats with high-fat diet, including triglyceride, total cholesterol and low-density lipoprotein content, and the efficacy of the extract can reach the same or better degree than that of a single compound of synephrine. Therefore, the high-content synephrine extract prepared according to the invention can be used for preparing health care products for weight control and blood fat reduction as active supplements.

Other embodiments

All features disclosed in this specification may be combined in any combination. Any feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the foregoing description, one skilled in the art can readily ascertain the essential characteristics of the described embodiments, and without departing from the scope thereof, can make various changes and modifications to the embodiments to adapt to various citrus fruits and application conditions. Accordingly, other embodiments are within the scope of the invention.

Claims

1. A process for preparing a synephrine-rich extract from unripe citrus fruit parts, said process comprising:

taking dried immature citrus fruit or pericarp thereof;

combining the extract liquor; and

2. The method of claim 1, wherein the immature citrus fruit is young fruit, fallen fruit or thinning fruit.

3. The method as claimed in claim 1, wherein the dried unripe citrus fruit or pericarp is ground prior to extraction.

4. The method of claim 1, wherein the part of the unripe citrus fruit is the exocarp of a citrus fruit in the young fruit stage.

5. The method of claim 1, wherein the ratio of the weight of the dried fruit or peel to the volume of methanol is 1:8 g/ml.

6. The method according to claim 1, wherein the ultrasonic method is ultrasonic oscillation at 4-25 ℃ for 20 minutes-2 hours.

7. The method of claim 1, wherein the stirring is carried out at an extraction temperature of 25-28 ℃ for 20 minutes to 2 hours.

8. The method of claim 1, wherein the microwave method comprises performing the extraction in a closed microwave-assisted extraction system at an elevated temperature of 30 ℃ to 64 ℃ for 10-15 minutes.

9. An extract with high content of synephrine isolated from immature citrus fruit parts, prepared by the method according to claim 1.

10. The synephrine-rich extract of claim 9, wherein the unripe citrus fruit part is the exocarp of a young fruit.

11. A composition for weight management comprising the high content synephrine extract of claim 9.

12. A composition for lowering blood lipid levels, comprising the synephrine-rich extract of claim 9.