Bone and joint protection composition and use thereof

Abstract

The invention relates to a bone and joint protection composition and use thereof. According to the composition, the four Chinese medicinal herbs Eucommiae Cortex, Achyranthis Bidentatae Radix, Astragali Radix and Chuanxiong Rhizoma are used, and the effects of enhancing the bone density, preventing and relieving osteoporosis, restraining the activity of osteoclast, promoting the function of bone cells, increasing the formation of calcified bone, promoting cartilage repair, delaying joint recession and repairing joint damage are achieved through multiple approaches and multiple levels. On the basis, by adding glucosamine hydrochloride, chondroitin sulfate and/or collagen, the bone density can be enhanced, the osteoporosis can be prevented and treated; the activity of the osteoclast can be restrained, the function of bone cells can be promoted, the formation of calcified bone can be increased, the cartilage repair can be promoted, joint recession can be delayed, and the joint damage can be repaired.

Claims

1. A bone and joint protection composition, characterized in that it consists of the following components by weight parts: TABLE-US-00007 Eucommiae Cortex 2-40 parts Astragali Radix 1-30 parts Achyranthis Bidentatae Radix 5-40 parts Chuanxiong Rhizoma 1-30 parts glucosamine hydrochloride 1-20 parts.

2. A bone and joint protection composition, characterized in that it consists of the following components by weight parts: TABLE-US-00008 Eucommiae Cortex 2-40 parts Astragali Radix 1-30 parts Achyranthis Bidentatae Radix 5-40 parts Chuanxiong Rhizoma 1-30 parts glucosamine hydrochloride 1-20 parts chondroitin sulfate 1-20 parts.

3. A bone and joint protection composition, characterized in that it consists of the following components by weight parts: TABLE-US-00009 Eucommiae Cortex 2-40 parts Astragali Radix 1-30 parts Achyranthis Bidentatae Radix 5-40 parts Chuanxiong Rhizoma 1-30 parts glucosamine hydrochloride 1-20 parts chondroitin sulfate 1-20 parts collagen 1-10 parts.

4. A method for increasing bone density and/or treating osteoporosis comprising administering to a subject in need thereof a composition according to claim 1.

5. A method for delaying joint recession, repairing joint damage, increasing the flexibility of bone and joint comprising administering to a subject in need thereof a composition according to claim 1.

Description

DETAILED DESCRIPTION

(1) The present invention discloses a composition and use thereof, and a person skilled in the art can realize the present invention by referring to the contents herein and modifying the process parameters appropriately. Of particular note is that all of the similar substitutions and modifications are obvious to a person skilled in the art, and they are deemed to be included in the present invention. The method and use of the present invention are described by means of the preferred Examples. The relevant persons can obviously realize and use the technology of the present invention by modifying or appropriately altering and combining the method and use described herein without departing from the content, spirit and scope of the present invention.

(2) The bone and joint protection composition provided by the present invention may be formulated into a variety of pharmaceutically acceptable dosage forms such as capsules, tablets, powders or granules and the like by conventional process. The present invention is not limited thereto.

(3) The raw materials and reagents used in the composition and use thereof provided by the present invention are commercially available.

(4) Hereinafter, the present invention is further illustrated by combining the Examples:

Example 1

(5) Weigh accurately Eucommiae Cortex 40 g, Achyranthis Bidentatae Radix 40 g, Astragali Radix 30 g, Chuanxiong Rhizoma 30 g, glucosamine hydrochloride 1 g, chondroitin sulfate 1 g, collagen 1 g, and mix them together.

Example 2

(6) Weigh accurately Eucommiae Cortex 30 g, Achyranthis Bidentatae Radix 30 g, Astragali Radix 25 g, Chuanxiong Rhizoma 10 g, glucosamine hydrochloride 2 g, chondroitin sulfate 2 g, collagen 2 g, and mix them together.

Example 3

(7) Weigh accurately Eucommiae Cortex 20 g, Achyranthis Bidentatae Radix 24 g, Astragali Radix 15 g, Chuanxiong Rhizoma 7 g, glucosamine hydrochloride 2 g, chondroitin sulfate 3 g, collagen 4 g, and mixm together.

Example 4

(8) Weigh accurately Eucommiae Cortex 21 g, Achyranthis Bidentatae Radix 40 g, Astragali Radix 20 g, Chuanxiong Rhizoma 10 g, glucosamine hydrochloride 14 g, chondroitin sulfate 10 g, collagen 20 g, and mix them together.

Example 5

(9) Weigh accurately Eucommiae Cortex 21 g, Achyranthis Bidentatae Radix 30 g, Astragali Radix 40 g, Chuanxiong Rhizoma 15 g, glucosamine hydrochloride 20 g, chondroitin sulfate 50 g, collagen 10 g, and mix them together.

Example 6

(10) Weigh accurately Eucommiae Cortex 2 g, Astragali Radix 30 g, Achyranthis Bidentatae Radix 5 g, Chuanxiong Rhizoma 30 g, glucosamine hydrochloride 1 g, chondroitin sulfate 10 g, collagen 10 g, and mix them together.

Example 7

(11) Weigh accurately Eucommiae Cortex 40 g, Astragali Radix 1 g, Achyranthis Bidentatae Radix 22 g, Chuanxiong Rhizoma 16 g, glucosamine hydrochloride 20 g, chondroitin sulfate 20 g, collagen 1 g, and mix them together.

Example 8

(12) Weigh accurately Eucommiae Cortex 20 g, Astragali Radix 16 g, Achyranthis Bidentatae Radix 40 g, Chuanxiong Rhizoma 1 g, glucosamine hydrochloride 11 g, chondroitin sulfate 1 g, collagen 5 g, and mix them together.

Example 9

(13) Weigh accurately Eucommiae Cortex 2 g, Astragali Radix 30 g, Achyranthis Bidentatae Radix 5 g, Chuanxiong Rhizoma 30 g, and mix them together.

Example 10

(14) Weigh accurately Eucommiae Cortex 40 g, Astragali Radix 1 g, Achyranthis Bidentatae Radix 22 g, Chuanxiong Rhizoma 16 g, and mix them together.

Example 11

(15) Weigh accurately Eucommiae Cortex 20 g, Astragali Radix 16 g, Achyranthis Bidentatae Radix 40 g, Chuanxiong Rhizoma 1 g, and mix them together.

Example 12

(16) Weigh accurately Eucommiae Cortex 2 g, Astragali Radix 30 g, Achyranthis Bidentatae Radix 5 g, Chuanxiong Rhizoma 30 g, glucosamine hydrochloride 1 g, and mix them together.

Example 13

(17) Weigh accurately Eucommiae Cortex 40 g, Astragali Radix 1 g, Achyranthis Bidentatae Radix 22 g, Chuanxiong Rhizoma 16 g, glucosamine hydrochloride 20 g, and mix them together.

Example 14

(18) Weigh accurately Eucommiae Cortex 20 g, Astragali Radix 16 g, Achyranthis Bidentatae Radix 40 g, Chuanxiong Rhizoma 1 g, glucosamine hydrochloride 11 g, and mix them together.

Example 15

(19) Weigh accurately Eucommiae Cortex 2 g, Astragali Radix 30 g, Achyranthis Bidentatae Radix 5 g, Chuanxiong Rhizoma 30 g, glucosamine hydrochloride 1 g, chondroitin sulfate 10 g, and mix them together.

Example 16

(20) Weigh accurately Eucommiae Cortex 40 g, Astragali Radix 1 g, Achyranthis Bidentatae Radix 22 g, Chuanxiong Rhizoma 16 g, glucosamine hydrochloride 20 g, chondroitin sulfate 20 g, and mix them together.

Example 17

(21) Weigh accurately Eucommiae Cortex 20 g, Astragali Radix 16 g, Achyranthis Bidentatae Radix 40 g, Chuanxiong Rhizoma 1 g, glucosamine hydrochloride 11 g, chondroitin sulfate 1 g, and mix them together.

Example 18 Comparative Example

Comparative Example 1

(22) Weighting Astragali Radix 30 g, Chuanxiong Rhizoma 40 g, glucosamine hydrochloride 1 g, chondroitin sulfate 1 g, collagen 1 g, and mix them together.

Comparative Example 2

(23) Weighting Epimedii Folium 3 g, Lycii Fructus 3 g, Astragali Radix 30 g, Chuanxiong Rhizoma 30 g, glucosamine hydrochloride 1 g, chondroitin sulfate 1 g, collagen 1 g, and mix them together.

(24) Bone and Joint Protection Test

Experiment 1 Animal Efficacy Test of Increasing Bone Density and Preventing Osteoporosis

(25) 1 Experimental Samples

(26) 1.1 Experimental group: the composition of the present invention: the composition provided by Example 9; batch number: 0816; dose recommended for adults: 2.88 g/d.

(27) Low-dose sample group: the amount by oral gavage for rats per day corresponds to 10 times of the dose recommended for adults (converted to dose per kg Bw), i.e., 0.048 g/100 g.

(28) High-dose sample group: the amount by oral gavage for rats per day corresponds to 30 times of the dose recommended for adults (converted to dose per kg Bw), i.e., 0.144 g/100 g.

(29) 1.2 The control group: Comparative Example samples: the composition of Comparative Example 1, Comparative Example 2; dose recommended for adults and the amount by oral gavage for rats are the same as those of the experimental group.

(30) Positive control and dose: estradiol valerate tablets, manufacturer: Bayer Healthcare Co., Ltd. (Guangzhou Branch), batch number: J20130009, specification: 0.1 g/tablet, the amount by oral gavage for rats per day is 0.1 mg/100 g.

(31) 2 Reagents and Instruments

(32) Main instruments: PUT electronic balance (Shenzhen Amput Electronic Technology Co. Ltd), mettler Toledo p1303, A4 atomic absorption spectrometer (Thermo Fisher Scientific Inc., USA).), DXA Prodigy Bone Densitometer (GE Lunar, USA), DHG-9053 Electric thermostatic air dry oven (Shanghai Sanfa Scientific Instrument Co., Ltd.); DZF-6020 vacuum drying oven (Shanghai keelrein instruments Co., Ltd.), Fixed cage, surgical instruments, syringes, etc.

(33) Main reagents: nitric acid (guarantee reagent, Guangzhou Chemical Reagent Factory); disodium ethylenediamine tetraacetate (Guangzhou Chemical Reagent Factory); calcium standard solution (1000 ug/ml 5% HCl medium), purchased from General Research Institute of China National Steel Material Test Center.

(34) 3 Experimental Animals

(35) SD rats in SPF grade, female, weeks of age at the time of purchasing: about 6 weeks old, weighing 220-250 g.

(36) 4 Statistical Processing of Experimental Results

(37) Analysis of variance was adopted: firstly, test of homogeneity of variances was conducted, and F value was calculated. F value<F.sub.0.05 indicates that there is no significant difference between the mean of each group; F valueF.sub.0.05, P0.05, a pairwise comparison method directed against the means of multiple experimental groups and one control group was used for conducting statistics; data of non-normality or uneven variance were subjected to appropriate variable conversion, and the data post conversion were subjected to statistics upon satisfying the requirements of normality or homogeneity of variance; if the requirements of normality or homogeneity of variance were still unsatisfied after variable conversion, rank sum test would be used for statistics.

(38) 5 Experimental Methods and Results

(39) 5.1 Experimental Method

(40) The methods in Technical specifications for inspection and evaluation of health food (2003 edition) was adopted. SD Rats weighing 220-250 g were adopted and maintained under normal feed, quarantine and observation in the experimental environment (barrier systems) for three days. The rats were anesthetized by intraperitoneal injection of 350 mg/kg BW of chloral hydrate solution. After abdominal fixation, the hairs at medioventral line which are 3-4 cm away from the vaginal opening were removed. The skin was sterilized with tincture of iodine and alcohol. Upon slight drying, the skin and abdominal muscle were incised by 2-3 cm. White fat was visible over the incision horizon. After poking away the fat layer, the uterus was found. The uterine horn at one side was gently pulled out; at its end, an ovary enveloped by fat mass was visible. Upon separation of fat mass, the ovary in pink or yellowish red color can be seen. The ovary was clipped by a hemostatic clamp, and the fallopian tube below the ovary (including fat) was ligated with a silk thread. The ovary was cut off (checking whether it was completely cut off), and the uterine horn was returned back to the abdominal cavity by the way. The ovary at the other side was cut off by the same method. After the suture of abdominal muscles and skin layer by layer, re-sterilization was conducted. Finally, 20,000 U penicillin was injected intramuscularly. Oophorectomy may also be operated via the incision at costovertebral angle of the back. To ensure successful operation, rats were subjected to vaginal smear check (a small amount of physiological saline was drawn with a pipette, which was gently inserted into the vagina for 1-2 cm; after rinsing for several times, the rinse was drawn off, coated on the slide and investigated under microscope) on the fifth day post the removal of the ovaries (once a day for consecutive 5 days) so as to check whether the ovaries of the rats were completely removed. The smears showing estrous reaction (a large number of semi-transparent, flat epidermal cells were seen under microscope) indicated that the ovaries of the animals were not completely removed; under such circumstances, the animals should be discarded. The sham operation group was composed of 12 rats that are not ovariectomized. The other trial drug groups, which were composed of rats that were screened post surgical operation to be successful in the oophorectomy, were observed for 3 days under normal conditions and then randomly divided into 8 groups (12 rats per group): model control group, positive control group, low-dose sample group, high-dose sample group. The sham operation group and model group were given distilled water per day by oral gavage, the rest of the sample groups were given corresponding doses of the test drug samples (one feeding per day for three months).

(41) 5.2 Observed Indicators

(42) 5.2.1 Observation of the General State

(43) Animals were observed daily for status (exterior signs, behaviors, fecal property, feeding conditions, etc.). And the animals were weighed one week after each feeding to observe the growth condition.

(44) 5.2.2 Determination of Bone Calcium

(45) 5.2.2.1 Sample Collection and Measurement of Bone Calcium

(46) The determination was conducted by means of atomic absorption flame photometry with the following parameters: the flow rate of the burning acetylene gas was 1.4 ml, the flame height was 8.6 cm, the detection wavelength was 422 nm; the femora of the rats at one side were dried to constant weight at a 105 C. oven, then weighted for dry weight; after weighting, the leg bones of the rats were dissolved with nitric acid and diluted to 10 ml, from which 0.10 ml was extracted, and diluted 700-fold with 0.02M ethylenediamine tetraacetic acid solution to subject to sample analysis.

(47) 5.2.2.2 Determination of Bone Density

(48) The femora at both sides were measured for bone density with a Prodigy DXA Bone Densitometer. The femora at both sides were positioned for measurement.

(49) 5.3 Experimental Results

(50) 5.3.1 Observation of the General State

(51) Compared with the sham operation group, animals from feeding and modeling groups had slightly less smooth hairs, but there was no significant difference in terms of other exterior signs, behaviors, fecal property, etc. Compared with the model control group, the test sample group, Comparative Example 1 group, Comparative Example 2 group and the positive control group did not have significant difference in terms of exterior signs, behaviors, fecal property, etc.

(52) As for the body weight, after feeding for 12 weeks, the body weights of animals from test sample, Comparative Example 1, Comparative Example 2 and the positive control groups were significantly higher than those of the sham operation group, but lower than those of the model group.

(53) 5.3.3 Effect on Bone Calcium Content and Bone Density of Left Femora in Rats

(54) TABLE-US-00005 TABLE 1 Effect on bone calcium content and bone density of left femora in rats (x s.d., N = 12) bone density bone calcium Groups BMD (g/cm.sup.3) content (mg/g) sham operation group 0.212 0.008 193.9 38.8 model group 0.195 0.010# 135.4 24.8# Control positive control group 0.207 0.010** 178.2 29.3** group Comparative Example 0.197 0.013 138.3 30.2 1 low-dose group Comparative Example 0.199 0.011 141.0 28.8 1 high-dose group Comparative Example 0.198 0.009 140.8 28.3 2 low-dose group Comparative Example 0.200 0.009* 145.1 19.9 2 high-dose group Experi- test sample (Example 0.202 0.011* 166.2 19.7* mental 9) low-dose group group test sample (Example 0.204 0.008* 167.3 20.4* 9) high-dose group Note: #denotes P < 0.01 versus the sham operation group; *denotes P < 0.05 versus the model group, **denotes P < 0.01 versus the model group.

(55) Table 1 shows that, compared with the sham operation group, the femoral bone density values and bone calcium contents of the model group were significantly decreased, suggesting that the rat osteoporosis model was successfully established.

(56) Compared with the model group, the bone density values of low-dose sample groups and high-dose sample group were significantly higher (P<0.05), and the bone calcium contents were also significantly increased (P<0.05). The bone density and bone calcium content values of positive control group were also significantly higher than those of the model group (P<0.01).

(57) Compared with the model group, the bone calcium contents and bone density values of Comparative Example 1 group have the rising trend, but without statistical difference (P>0.05). The bone density values of Comparative Example 2 high-dose group were significantly increased (P<0.05), but the bone calcium contents only had the rising trend (P>0.05).

(58) Compared with the positive control group, the femoral bone density values and bone calcium contents of test sample high-dose group and low-dose group were significantly decreased (P<0.01); compared with Comparative Example 1 high- and low-dose groups, both of the femoral bone density values and bone calcium contents of test sample high-dose group and low-dose group were significantly increased (P<0.05); compared with Comparative Example 2 low-dose group, both of the femoral bone density values and bone calcium contents of test sample high- and low-dose groups were significantly increased (P<0.05); compared with Comparative Example 2 high-dose group, both of the femoral bone density values and bone calcium contents of test sample high-dose group were significantly increased (P<0.05).

(59) The above data showed that, the samples have the effects of elevating bone calcium content and increasing bone density, and can be used to relieve osteoporosis, whereas Comparative Example 1 and Comparative Example 2 do not have obvious effect of relieving osteoporosis.

(60) The effect of relieving osteoporosis of high-dose sample groups is superior to that of Comparative Example 1 high-dose group and low-dose group and Comparative Example 2 high-dose group and low-dose group. The effect of relieving osteoporosis of low-dose sample groups is superior to that of Comparative Example 1 high-dose group and low-dose group and Comparative Example 2 low-dose group. The compositions prepared by Examples 1 to 8, Examples 10 to 17 were subjected to the above experiments, and the experimental results were the same or similar as the effects of the composition prepared by Example 9 and were without significant difference (P>0.05), which shows that all of the compositions prepared by Examples 1 to 17 of the present invention have the functional effect of increasing bone density.

Experiment 2 Animal Efficacy Test of Repairing Joints

(61) 1 Experimental Samples

(62) 1.1 Experimental group: the composition of the present invention: the composition provided by Example 9; batch number: 0816; dose recommended for adults: 2.88 g/d.

(63) Sample Dose: the amount by oral gavage for New Zealand white rabbits per day corresponds to 30 times of the dose recommended for adults (converted to dose per kg Bw), i.e., 0.144 g/100 g.

(64) 1.2 The control group: Comparative Example samples: the composition of Comparative Example 1, Comparative Example 2; dose recommended for adults and the New Zealand rabbits are the same as those of the experimental group.

(65) Normal control group: the non-surgical group was given distilled water at same amount as other groups.

(66) 2 Reagents and Experimental Instruments

(67) Experimental instruments: Olympus optical microscope camera system (BH-2 type); German ZEISS (Zeiss) Axiotron research microscope.

(68) Main reagents: EDTA (Chengdu Chemical Reagent Company); paraformaldehyde (Beijing Chemical Reagent Company); AB-PAS staining reagent; papain (Sigma-Aldrich Co. LLC.) and so on.

(69) 3 Experimental Animals

(70) Healthy New Zealand rabbits, weighing 2.5-3.0 Kg, male, fed on standard diet, accessible to food and water freely.

(71) 4 Statistical Processing of Experimental Results

(72) Analysis of variance was adopted: firstly, test of homogeneity of variances was conducted, and F value was calculated. F value<F.sub.0.05 indicates that there is no significant difference between the mean of each group; F valueF.sub.0.05, P0.05, a pairwise comparison method directed against the means of multiple experimental groups and one control group was used for conducting statistics; data of non-normality or uneven variance were subjected to appropriate variable conversion, and the data post conversion were subjected to statistics upon satisfying the requirements of normality or homogeneity of variance; if the requirements of normality or homogeneity of variance were still unsatisfied after variable conversion, rank sum test would be used for statistics.

(73) 5 Experimental Method and Results

(74) 5.1 Experimental Methods

(75) According to the method disclosed in literature, the bone and joint disease model was established by injecting papain into the joint cavity of the rabbits' knees. After the rabbits were anesthetized with sodium pentobarbital (30 mg/kg, intravenous injection), the hairs around the knee joint of the left hind leg were shaved. The knee joint was slightly bent, and then 0.3 ml of 4% papain solution in physiological saline was injected into the rabbits' knee joint cavity through patellar ligament (injected at both sides of the joint) for once every 3 days. The injections were implemented for consecutive 3 times (note: New Zealand rabbits from normal control group do not need to have this operation). Two weeks after the completion of the last injection of papain, the administration of samples were started. The samples were administrated for consecutive 6 weeks (the model group and normal control group were consecutively administered with distilled water), the rabbits in each group were all sacrificed, then the knee joints from the normal control group and the inner femora at the left knee joints from the rest of modeling groups were cut off by means of a sharp blade to generally observe the conditions of synovium and cartilage by naked eyes. Using cartilage AB-PAS staining, the morphological structure of articular cartilage and change of coloration of proteoglycan in cartilage matrix were observed under light microscope. Using modified histologyhistochemical grading system (HHGS), the articular cartilages from each of the specimens were subjected to pathological grading and scoring.

(76) 5.2 Observed Indicators

(77) 5.2.1 Observation of the General State

(78) Animals were observed daily for status (exterior signs, behaviors, fecal property, feeding conditions, etc.). And the animals were weighed weekly.

(79) 5.2.2 Observation of the Cartilage

(80) 5.3 Experimental Results

(81) 5.3.1 Observation of the General State

(82) During the duration of the experiment, it was seen that the New Zealand rabbits from the normal group had good mental state, and had no abnormal changes in hair color. However, the New Zealand rabbits from each of the modeling groups had claudication in varying degrees and significantly reduced spontaneous activities as well as significantly affected knee joint movements.

(83) As for the body weight, after feeding for 12 weeks, the body weights of animals from Comparative Example 1, Comparative Example 2 and the positive control groups were significantly lower than those of the normal control group, but higher than those of the model group.

(84) 5.3.2 Observation of the Cartilage

(85) The appearance of the joint cartilages of rabbits' knees from the normal group was in bright blue-white color; was free from cracks, softening or defects; and was rigid upon touch. The amount of synovial fluid was small, and the texture of the synovial fluid was clear and transparent. 8 weeks after modeling, the knee joints of each group were injured seriously. The manifestations included: the faces of the joint cartilage were significantly uneven, or even the cartilages had intermittent cracks and defects. Among these, the cartilage situations of the model group were more serious than the other groups.

(86) Upon AB-PAS staining, the acidic mucopolysaccharides in cartilage matrix were in blue color, whereas the neutral mucilage substances were in red color. The AB-PAS staining of normal group showed that the cartilage staining was clear and uniform and was without the phenomenon of loss of staining; the chondrocytes were distributed evenly and without the clustering of chondrocytes; the surface layer of the cartilage was smooth and flat. 8 weeks after modeling, it could be seen that the surface layer of the cartilage from the model group had the phenomenon of loss of staining, the staining was not uniform, and the surface of the cartilage was uneven. It could also be seen that, as for the rest of the various sample groups, the cartilage staining was uneven and ambiguous, the morphology of the chondrocytes and matrix was unclear, and the surface layer and middle layer or even the deep layer had a large amount of unstained area, concurrently with varying degrees of cartilage cracks or defects. The scoring are as follows:

(87) TABLE-US-00006 TABLE 2 The comparative table for AB-PAS staining pathological scores (x s.d., N = 6) groups AB-PAS scores Control group Model group 11.30 0.91# Normal control 0.55 0.61 group Comparative 10.77 1.07 Example 1 group Comparative 10.38 0.95 Example 2 group Experimental group test sample group 8.85 0.81* Notes: #denotes P < 0.05 versus normal control group; *denotes P < 0.05 versus model group.

(88) Compared with the model group, the pathological scores of the experimental group were decreased compared with the model group (P<0.05); Comparative Example 1 and Comparative Example 2 groups had the descending trend but no significant difference (P>0.05). Compared with the normal control group, Comparative Example 1 group and Comparative Example 2 group, the experimental group had a significant difference (P<0.05). This fact indicates that the experimental group can elevate the proteoglycan content of cartilage matrix and promote joint repair, but the Comparative Example 1 group and Comparative Example 2 group do not have this effect. Moreover, the effect of the experimental group is superior to those of the Comparative Example 1 group and Comparative Example 2 group.

(89) The compositions prepared by Examples 1 to 8, Examples 10 to 17 were subjected to the above experiments, and the experimental results were the same or similar as the effects of the composition prepared by Example 9 and were without significant difference (P>0.05), which shows that all of the compositions prepared by Examples 1 to 17 of the present invention have the functional effect of improving joint movement capacities.

(90) The above are only preferred embodiments of the present invention, and it should be noted that, a person having ordinary skill in the art can also make improvements and modifications thereto without departing from the principles of the present invention, and these improvements and modifications should also be considered as falling within the scope of the present invention.