Traditional Chinese medicine compound for postpartum recovery of weaned female yaks as well as preparation method and application of traditional Chinese medicine compound

Abstract

The invention discloses a traditional Chinese medicine compound for recovering body functions of postpartum weaned female yaks and improving immunity as well as a preparation method and application thereof, and belongs to the technical field of medicines. The traditional Chinese medicine compound (TCMC3) is prepared from ramie roots, ligusticum wallichii, prepared rehmannia roots, radix paeoniae alba, astragalus membranaceus, Tibetan codonopsis pilosula, atractylodes macrocephala koidz, fructus aurantii, pericarpium citri reticulatae, scutellaria baicalensis, perilla frutescens, artemisia inflata and liquorice. After the traditional Chinese medicine compound disclosed by the invention is applied to postpartum weaned female yaks, the physique and body functions of the postpartum weaned female yaks can be enhanced, the reproductive performance of the postpartum weaned female yaks can be guaranteed, and the antioxidant capacity, immunity and the like of the postpartum weaned female yaks can be improved, so that the effects of recovering the physique and body functions of the postpartum weaned female yaks, enhancing the immunity, promoting oestrus and the like are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a traditional Chinese medicine compound for postpartum recovery of weaned female yaks, a preparation method thereof, and an application thereof. Background Art

[0002] Yaks, known as the "boats of the plateau", are rare species mainly living in high-altitude areas. The number of yaks in China has exceeded 10 million heads, accounting for more than 94% of the total number of yaks on the entire earth. Due to the special physiological characteristics of yaks, they can well adapt to the high-altitude environment. Moreover, in the case of low air pressure and hypoxia, they can more effectively carry out blood flow oxygen transport and material exchange to maintain normal life activities. As the only large mammal on the Qinghai-Tibet Plateau, yaks not only provide meat, milk, etc. for local herdsmen, but also are used as tools for trade and transportation. The cowhide, cow hair, cow cashmere and cow horns of yaks all have high economic value, providing living necessities and economic support for plateau herdsmen. Not only that, the feces of yaks can also be used as fuel for herdsmen to make fire and survive. Therefore, yaks play an important role in the production, life and economic sources of plateau pastoral residents, and can be described as "treasure livestock".

[0003] Yaks are seasonal estrus animals. The estrus season is generally from June to November, the gestation period is 250-260 days, calving mostly occurs from March to May of the following year, and the age of first estrus of female yaks is from two and a half to four and a half years old, among which three and a half years old accounts for a relatively large proportion, but the calving rate is as low as 20%, and the survival rate of calves is also relatively low. The reproductive rate of adult female yaks is mostly one calf every two years, or even one calf every three years. Since the pregnancy stage of female yaks (about 250-260 days) is relatively long, most of the energy stored in the body will be consumed in large quantities, resulting in the body condition of female yaks being difficult to recover in a timely and effective manner for a long time after giving birth. The estrus rate of female yaks after calving in the estrus season of the same year is only 3%; moreover, the limitedness of forage resources also restricts the nutritional intake of weaned female yaks after giving birth, thereby leading to the prolongation of the anestrus period after giving birth in female yaks, resulting in most female yaks showing one calf every two years, two calves every three years, or even one calf every three years, with low reproductive efficiency, which severely restricts the production efficiency of yaks on the Qinghai-Tibet Plateau. In addition, due to factors such as extensive management, incomplete infrastructure, prominent grass-livestock contradictions, and degradation of excellent breeds, the impact on weaned female yaks after giving birth is aggravated, and at the same time, the sustainable development of the yak industry is also severely restricted.

[0004] Therefore, in order to meet the breeding needs of the yak industry in China, developing a drug that is efficient, safe, not prone to drug resistance, and can restore the body functions of weaned female yaks after giving birth, ensure their reproductive performance, and enhance immunity is one of the problems that need to be urgently solved by those skilled in the art. Summary of the Invention

[0005] In view of the problems of the above-mentioned existing technologies, the present invention provides a traditional Chinese medicine compound capable of restoring the body functions of postpartum weaned female yaks and enhancing their immunity, as well as its preparation method and application. After the traditional Chinese medicine compound disclosed by the present invention is applied to postpartum weaned female yaks, it can enhance the physique of postpartum weaned female yaks, significantly improve the antioxidant capacity of postpartum weaned female yaks, etc., so as to achieve the effects of restoring the body functions of postpartum weaned female yaks and enhancing their immunity.

[0006] Compared with existing drugs, the present invention has the following beneficial effects:

[0007] (1) The traditional Chinese medicine compound (TCMC3) provided by the present invention is added to the feed for supplementary feeding of postpartum weaned female yaks. Through experiments, it is found that: postpartum weaned female yaks fed with the feed containing the traditional Chinese medicine compound can improve their antioxidant capacity (such as Figure 1 ); regulate the composition structure of their rumen microorganisms (such as Figure 5 ); and promote the restoration of body functions of postpartum weaned female yaks, enhance their physique and immunity.

[0008] (2) The traditional Chinese medicine compound (TCMC3) provided by the present invention is added to the feed for supplementary feeding of postpartum weaned female yaks. Through experiments, it is found that: the traditional Chinese medicine compound can reduce harmful bacteria by enriching beneficial bacteria, regulate the composition structure of the intestinal flora, promote the change of the intestinal flora of postpartum weaned female yaks, especially the increase in the relative abundance of beneficial bacteria and the decrease in the relative abundance of harmful bacteria (such as Figure 10 ); the traditional Chinese medicine compound provided by the present invention is safe, harmless, green and environmentally friendly. After being applied to postpartum weaned female yaks, it can enhance the physique and body functions of postpartum weaned female yaks, ensure their reproductive performance, improve the antioxidant capacity and immunity of postpartum weaned female yaks, and provide a new reference for the breeding of postpartum weaned female yaks in plateau areas. Description of the Drawings

[0009] Figure 1 Detection of serum indexes of postpartum weaned female yaks by the traditional Chinese medicine compound (TCMC3) provided by the present invention on the 15th day and the 30th day of the experiment;

[0010] Figure 2 Analysis of the α-diversity of rumen microorganisms of postpartum weaned female yaks by the traditional Chinese medicine compound (TCMC3) provided by the present invention on the 15th day and the 30th day of the experiment;

[0011] Figure 3 Venn diagram of rumen microorganisms of postpartum weaned female yaks by the traditional Chinese medicine compound (TCMC3) provided by the present invention on the 15th day and the 30th day of the experiment;

[0012] Figure 4For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the beta diversity analysis of the rumen microorganisms of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0013] Figure 5 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the effects on the rumen microorganisms of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0014] Figure 6 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the LEfSe analysis of the rumen microorganisms of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0015] Figure 7 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the alpha diversity analysis of the intestinal flora of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0016] Figure 8 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the Venn diagram of the intestinal flora of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0017] Figure 9 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the beta diversity analysis of the intestinal flora of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0018] Figure 10 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the effects on the intestinal flora of postpartum weaned female yaks on the 15th and 30th days of the experiment;

[0019] Figure 11 For the traditional Chinese medicine compound (TCMC3) provided by the present invention, the LEfSe analysis of the intestinal flora of postpartum weaned female yaks on the 15th and 30th days of the experiment. Detailed implementation manners

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0021] Embodiment 1

[0022] In this embodiment, the preparation method of the traditional Chinese medicine compound (TCMC3) is as follows:

[0023] (1) Weigh 50 g of ramie root, 50 g of chuanxiong rhizome, 50 g of rehmannia root, 30 g of white peony root, 30 g of astragalus root, 40 g of Codonopsis pilosula var. modesta, 60 g of atractylodes rhizome, 30 g of aurantii fructus immaturus, 30 g of tangerine peel, 30 g of scutellaria root, 30 g of perilla leaf, 20 g of Artemisia princeps, and 20 g of licorice root respectively according to the mass ratio of TCMC3.

[0024] (2) Dry and crush each of the herbs weighed in step (1) and then mix them to prepare the traditional Chinese medicine compound TCMC3 for storage and standby. This is the traditional Chinese medicine compound that can restore the body functions of postpartum weaned female yaks and enhance their immunity.

[0025] Example 2

[0026] This example is an experiment on the effects of the traditional Chinese medicine compound (TCMC3) on the serum indexes and rumen microbial diversity of postpartum weaned female yaks. Add the traditional Chinese medicine compound (TCMC3) prepared in Example 1 above to the basal diet for the experiment on postpartum weaned female yaks. Add the traditional Chinese medicine compound (TCMC3) to the basal diet at a ratio of 5%. In addition, environmental conditions such as enclosure, lighting, and ventilation are kept consistent in all groups.

[0027] Twelve postpartum weaned female yaks 80±15 days after parturition were selected and randomly divided into a control group and a traditional Chinese medicine compound (TCMC3) supplementary feeding group. n is the number of animals. DG group (traditional Chinese medicine compound supplementary feeding group) (n = 6): Feed 5 kg of basal diet supplemented with 5% of TCMC3 prepared in Example 1 every day. DH group (blank control group) (n = 6): Feed 5 kg of basal diet every day. Seven days before the experiment, administer 1 / 4 of the experimental dose from the 1st to the 4th day and 1 / 2 of the experimental dose from the 5th to the 7th day, and observe the status of female yaks for any adverse reactions. Starting from the 8th day of the experiment, administer the dose (5% traditional Chinese medicine compound), and the experimental period is 30 days. The feeding time is 9:30 and 15:00 every day to ensure that the traditional Chinese medicine compound is fully mixed with the basal diet, and free drinking water is provided during the experiment. On the 15th and 30th days of the experiment, collect the blood of all female yaks. The experimental yaks are all bled by jugular vein puncture. After the blood is left standing at room temperature for 2-3 h, centrifuge at 3500 r / min for 15 min to separate the serum for detecting the immune capacity and antioxidant capacity of postpartum weaned female yaks (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), malondialdehyde (MDA), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD)). Before the morning feeding on the 15th and 30th days of the experiment, in the DG group and DH group respectively, use an oral collection catheter to draw 50 mL of rumen fluid from the rumen of each female yak in each group. (Classified as DG1 (divided into DC1-DC6 between groups) and DH1 (divided into DD1-DD6 between groups) on the 15th day; classified as DG2 (divided into DC7-DC12 between groups) and DH2 (divided into DD7-DD12 between groups) on the 30th day) After filtering with 4 layers of sterilized gauze, it is subpackaged for subsequent 16S rRNA sequencing analysis.

[0028] As Figure 1 shown: On the 15th and 30th days of the experiment, there were significant differences in the antioxidant-related indexes SOD, T-AOC, GSH-Px, and MDA between the DG group and the DH group ( Figure 1 A-D), indicating that the traditional Chinese medicine compound can improve the antioxidant capacity of postpartum weaned female yaks; on the 15th and 30th days of the experiment, there were also significant differences in the inflammation-related indexes ( Figure 1 E-H), indicating that the traditional Chinese medicine compound reduced the occurrence of inflammation in postpartum weaned female yaks. As Figure 2As shown, the species accumulation curve used to measure and predict the increase in species richness with the increase in sample size shows a trend of flattening, indicating that the sequencing depth is sufficient to cover all species; while the Pielou_e index (p = 0.0004), Shannon index (p = 0.0043), and Simpson index (p = 0.031) of the DG group and the DH group are significantly different, indicating that the richness, diversity, evenness, and coverage of the DH group are higher ( Figure 2 A,B,C). As Figure 3 shown, the ASVs of the DG2 group are higher than those of the DH2 group. Among them, there are 3333 ASVs in common between DG1 and DH1, 3438 ASVs in common between DG2 and DH2, 10454 unique ASVs in DG1, 12085 unique ASVs in DG2, 11814 unique ASVs in DH1, and 11565 unique ASVs in DH2, indicating that the addition of the traditional Chinese medicine compound (TCMC3) results in significant differences in the rumen microbial composition ( Figure 3 A,B). As Figure 4 shown, on the 15th and 30th days of the experiment, the points of the DG group and the DH group are significantly separated, indicating that there are differences in the composition of the rumen microbial community of postpartum weaned female yaks after feeding the traditional Chinese medicine compound (TCMC3); and the stress value (<0.2) of the NMDS analysis also provides reliable results for further research and analysis ( Figure 4 A,B). As Figure 5 shown, on the 15th and 30th days of the experiment, the top three compositions of the relative abundances of rumen microorganisms in each group at the genus level are roughly the same, but there are certain differences in the relative abundances; on the 15th day of the experiment, the dominant genera in the DG1 group and the DH1 group are Limimorpha, SFMI01, Prevotella, and Cryptobacteroides. On the 30th day of the experiment, the dominant genera in the DG2 group and the DH2 group are the same as those on the 15th day of the experiment, but their relative abundances have changed slightly. Among them, the relative abundance of Prevotella in the DG group has increased significantly, while on the 30th day of the experiment, the relative abundance of the SFMI01 genus in the DG group has decreased significantly ( Figure 5 A); and from each individual, on the 15th day of the experiment, the relative abundance of the Limimorpha genus in the DG group has increased, on the 30th day of the experiment, the relative abundance of Prevotella in the DG group has increased significantly, and the relative abundance of the Limimorpha genus in the DH group has increased ( Figure 5 B), indicating that the addition of the traditional Chinese medicine compound (TCMC3) results in significant differences in the rumen microbial composition. As Figure 6As shown, according to LEfSe analysis, there are differences in the composition and structure of rumen microorganisms in the DG1 group, DH1 group, and DH2 group, with significantly different phyla and genera; the biomarker of the DG2 group is Bacteroidales, the biomarkers of the DH1 group are Clostridia and Firmicutes, and the biomarker of the DH2 group is Firmicutes.

[0029] Example 3

[0030] This example is an experiment on the effect of traditional Chinese medicine compound (TCMC3) on the intestinal flora of postpartum weaned female yaks. The traditional Chinese medicine compound (TCMC3) prepared in Example 1 above was added to the basal diet for the experiment on postpartum weaned female yaks. The traditional Chinese medicine compound (TCMC3) was added to the basal diet at a ratio of 5%, and the environmental conditions, such as enclosure, lighting, and ventilation, were kept consistent in all groups.

[0031] Twelve postpartum weaned female yaks 80 ± 15 days after parturition were selected and randomly divided into a control group and a traditional Chinese medicine compound supplementary feeding group, where n is the number of animals; DG group (traditional Chinese medicine compound supplementary feeding group) (n = 6): fed 5 kg of basal diet supplemented with 5% of TCMC3 prepared in Example 1 every day; DH group (blank control group) (n = 6): fed 5 kg of basal diet every day. Seven days before the experiment, 1 / 4 of the test dose was administered on days 1 - 4, and 1 / 2 of the test dose was administered on days 5 - 7, and any adverse reactions were observed. Starting from day 8 of the experiment, the dosing dose (5% traditional Chinese medicine compound) was given for a 30 - day experimental period. The feeding time was 9:30 and 15:00 every day to ensure that the traditional Chinese medicine compound was fully mixed with the basal diet, and free drinking water was provided during the experiment. On day 15 of the experiment, 200 g of fresh rectal feces of postpartum weaned female yaks were collected in the DG group and DH group respectively, and classified as DG1 (divided into DC1 - DC6 between groups) and DH1 (divided into DD1 - DD6 between groups); on day 30 of the experiment, 200 g of fresh rectal feces of postpartum weaned female yaks were collected in the DG group and DH group respectively, and classified as DG2 (divided into DC7 - DC12 between groups) and DH2 (divided into DD7 - DD12 between groups). The collected fresh fecal samples were used for 16S rRNA high - throughput sequencing to analyze the effect on the intestinal flora of postpartum weaned female yaks.

[0032] As Figure 7As shown, the species accumulation curve used to measure and predict the increase in species richness with the increase in sample size tends to flatten, and the Good's coverage index of each group is close to 100%, indicating that the sequencing depth is sufficient to cover all species. On the 15th day and 30th day of the experiment, there were no significant differences in the Simpson index, Shannon index, Pielou_e index, and Faith_pd index between the DG group and the DH group (p > 0.05), but there were significant differences in the Chao 1 index (p = 0.022), Goods_coverage index (p = 0.035), and Observed_cpecies index (p = 0.037), indicating that there were significant differences in species richness between the two groups, and the species richness of the DG group was higher ( Figure 7 A, B, C). As Figure 8 shown, there were 2571 common ASVs in the DG1 and DH1 groups, among which the DG1 group and the DH1 group had 5816 and 6224 unique ASVs respectively; there were 2404 ASVs in the DG2 and DH2 groups, among which the DG2 group and the DH2 group had 5204 and 5663 unique ASVs respectively ( Figure 8 A, B). As Figure 9 shown, the points of the DG group and the DH group showed obvious separation to varying degrees, indicating that the intestinal flora structure of postpartum weaned female yaks changed after feeding with the traditional Chinese medicine compound (TCMC3); the stress value (<0.2) of the NMDS analysis also provided reliable results for further research and analysis ( Figure 9 A, B). As Figure 10 shown, at the genus level, the top three compositions of the relative abundances of the intestinal flora in each group were roughly the same on the 15th day and 30th day, but there were certain differences in the relative abundances; on the 15th day of the experiment, Faecousia, Cryptobacteroides, and Arthrobacter_B in the DG group and the DH group were the three most common genera, and these three dominant genera were also present on the 30th day of the experiment, but their relative abundances changed slightly; among them, on the 30th day of the experiment in the DG group, the relative abundance of Faecousia decreased, while the relative abundance of Arthrobacter_B increased significantly ( Figure 10 A); from each individual, on the 15th day of the experiment, the relative abundances of Faecousia and Arthrobacter_B in the DG group increased, and on the 30th day of the experiment, the relative abundance of Cryptobacteroides in the DG group decreased ( Figure 10 B). As Figure 11As shown, according to LEfSe analysis, there are differences in the composition and structure of the intestinal flora in the DG1 group, DG2 group, and DH1 group, with significantly different phyla and genera; the biomarker of the DG1 group is f_Bacteroidaceae, and significant enrichments of p_Actinobacteriota, f_Micrococcaceae, o_Actinomycetales, c_Actinomycetia, etc. are detected in the DG2 group, and significant enrichments of c_Bacilli, p_Firmicute_D, etc. are detected in the DH1 group( Figure 11 ).

[0033] The above introduces the traditional Chinese medicine compound for restoring the body functions of postpartum weaned female yaks, enhancing immunity, its preparation method and application. The application examples in this article elaborate on the principle and implementation mode of the present invention. The description of the above examples is only used to help understand the method and core idea of the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A traditional Chinese medicine compound (TCMC3) for restoring the body functions of postpartum weaned female yaks and enhancing immunity, characterized in that, The raw materials of the traditional Chinese medicine compound are all traditional Chinese medicines in China, with relatively small side effects.

2. The preparation method of a traditional Chinese medicine compound for restoring the body functions of postpartum weaned female yaks and enhancing immunity according to claim 1, characterized in that, The specific steps are as follows: (1) Weigh according to the mass ratio of each medicinal material in the traditional Chinese medicine compound. TCMC3: 50 g of ramie root, 50 g of chuanxiong rhizome, 50 g of prepared rehmannia root, 30 g of white peony root, 30 g of astragalus root, 40 g of Codonopsis thalictrifolia Wall., 60 g of atractylodes rhizome, 30 g of fructus aurantii, 30 g of dried tangerine peel, 30 g of scutellaria baicalensis, 30 g of perilla, 20 g of Artemisia princeps Pamp., 20 g of liquorice root; (2) After drying and pulverizing each herb in the pharmaceutical composition obtained in step (1), mix them in an appropriate proportion to make a Chinese herbal medicine, and store it for later use, which is a traditional Chinese medicine compound capable of restoring the body function of postpartum weaned female yaks and enhancing immunity.

3. Use of the traditional Chinese medicine compound for restoring the body function of postpartum weaned female yaks and enhancing immunity according to claim 2 in the preparation of drugs or feed additives for postpartum weaned female yaks.

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