Animal feed compositions and uses of triterpenoid saponin obtained from Camellia L. plants

An animal feed composition is disclosed which comprises triterpenoid saponin obtained from Camellia L. plants and having the properties of improving immune function, enhancing antibacterium and antivirus activities, antimutation, antioxidation and scavenging free radicals in human beings and animals.

TECHNICAL FIELD 
The present invention relates to a method of preventing animal diseases and 
promoting animal growth and development. In particular, the method of the 
present invention comprises extracting bioactive ingredients from oil 
plants and adding them into feed and food as a feed additive and nutrient, 
respectively, to improve the immune function of animals and to promote the 
growth and developement of animals. 
BACKGROUND ART 
Plants belonging to Camellia L. are very popular in China, Japan, India and 
many other South-East Asian countries. Among of them, plants from three 
species C. sinensis, C. oleifera and C. japonica, are of significant 
economic value. For example, leaves of C. sinensis plants can be used to 
produce tea and seeds of C. oleifera plants can be used to produce edible 
oil, while C. japonica plants are useful as ornamental plants. 
With the development of the modern feed industry, a number of antibiotics 
have been used as feed additive to prevent animal diseases and to promote 
animal growth. In 1990, while about 2000 tons of antibiotics were used as 
feed additives in China, about 1350 tons and 1300 tons of antibiotics were 
used in West Europe and Japan, respectively, in poultry and livestock 
feed. Recently, sale value of antibiotics reached US $1.019 billions in 
the USA and utilization of antibiotics is increasing by 3% annually world 
wide. Long-term utilization of antibiotics, however, can result in 
drug-resistance of microbes which will create world-wide environment 
pollution via R-factor translocation, and residual problem in animal 
products which will be a that to human health. The WHO (World Health 
Organization) and the FAO (Food and Agriculture Organization, United 
Nations) are taking great consideration about research on alternatives of 
antibiotics as feed additives. Currently, research and development on 
natural material as a feed additive has made a great deal of progress. For 
example, feed microbes and traditional Chinese medicines have been used 
extensively. However, feed microbes are prone to becoming inactive during 
feed processing and can only replace antibiotics partially. Traditional 
Chinese medicine additives can not be used extensively, due to their 
sophistication in composition and instability in function. Therefore, 
there is a necessity to (1) use natural bioactive ingredients as feed 
additive to replace antibiotics, (2) exclude the residue of antibiotics 
residue in animal products, (3) improve product quality, and (4) alleviate 
environmental pollution. 
S. Aoyama first separated thea saponin from tea seed cake in 1931. (Journal 
of Pharmacology, 51(5): 367, 1931). However he did not obtained this pure 
chemical. In 1952, M. Ishidate and Y. Ueda of Tokyo University obtained 
the pure crystals of thea saponin. (Ishidate, M. and Ueda, Y., Journal of 
Pharmacology, 72 (11): 1525, 1952.). From the 1970's, a series of studies 
on separation, characterization and utilization of thea saponin were 
conducted in major tea-producing countries. Many extracting methods and 
products had been developed during this period. 
It is now clear that saponin obtained from Camellia L. belongs to 
triterpenoid compounds. It is a group of complicated compounds composed of 
aglycone (C.sub.30 H.sub.50 O.sub.6), sugars and organic acids. The 
chemical structures of saponin obtained from Camellia L. have been 
disclosed in many references. For example, in I. Yoshioka et al., Chemical 
Pharmacology Bulletin (Tokyo) 1970, 18. 1610; Yoshioka, et al., Thea 
sapogenol A., The Major Sapogenol of the Seeds Saponin of Thea"; Sinensis 
L., Tetrahedron Letters, 1966 (48): 5979-5984, 5973-5978; and I. Yoshioka, 
et al., Saponin and Sapogenol III Seeds Sapogenols of Thea Sinensis L (3), 
Thea Sapogenol E and Minor Sapogenols, Chem. Phar. Bull. 1971, 19 (6) 
1186-1199. 
Development in thea saponin research offers great opportunity for complete 
utilization of by-products of tea seed and tea leaves. In 1972, G. R. 
Roberts and his colleague (G. R. Roberts et al., Tea QUARTERLY 43 (3), 
1972) from Sri Lanka improved thea saponin extracting technology, 
industrialized its preparation, and suggested several other methods to use 
tea seed cake from Camellia plants, (R. L. Wickremastinghe et al., 1972. 
Tea Quarterly 43 (3)). As estimated by T. Yaziciglu and his colleague, 
about 600 tons of thea saponin could be extracted from 15,000 tons of tea 
seed in Turkey. So far, industrialization of thea saponin production has 
been possible, and Nippon Isome Grease Chemical Co. is already 
commercializing the production of thea saponin. 
Saponin obtained from Camellia L. has extensive utilization in industry. 
Its utilization in medicine was the earliest research field. However, this 
field developed slowly though there are many pharmacologists investigating 
the problem. Pharmacological effects of the saponin include antiosmosis, 
antiphlogistics, and control of coughing. It was reported that the saponin 
has special effect on many kinds of oedema (dropsy). 
Industry utilization of saponin obtained from Camellia L. is a newly 
developed field. It could be used to produce kinds of water and oil 
emulsion, preservatives, foaming agent in the beer industry and detergents 
in daily industry. It could maintain the color of fabrics as it resists 
damage to dye on the fabrics. When used in laundering process, it prevents 
shrinking of woolen products and maintains the luster of fabrics as well. 
The saponin can also be used in photographic industry. 
Saponin obtained from Camellia has extensive usage in agriculture, 
expecially as insecticide, germicide and binding agent in spray pesticide. 
Its major benefit is to avoid pesticide residue and protect environment. 
However, research on the utilization of saponin obtained from Camellia as 
feed additive to replace antibiotics has not been documented yet, nor have 
the effects of said saponin on animal growth and immune function 
promotion. 
After research and development of saponin obtained from Camellia L. for 
many years, the present inventor has unexpectedly found that saponins 
extracted from the seed cake of Camellia L. could improve immune function 
and has the added beneficial effects of being anti-bacterial and 
anti-viral. It is, in fact, safe for animal consumption under some 
conditions and can enhance the growth of the animals. 
It is one object of the present invention to provide a method of promoting 
the growth of animals. 
It is another object of the present invention to provide a method of 
improving immune function and anti-bacterial and anti-viral effects. 
It is another object of the present invention to provide a use of the 
bioactive extract of oil plants as feed additive. 
It is another object of the present invention to provide a novel health 
care agent. 
DETAILED DESCRIPTION OF THE INVENTION 
As used in the context of the present invention, the term "seed cake" 
refers to residues obtained from seeds of oil-bearing plants after the 
extraction of oil therefrom. 
The term "Camellia seed cake" refers to seed cake obtained from Camellia L. 
plants, such as from C. sinensis, C. oleifera and C. japonica plants. 
Triterpenoid saponin used in this invention was extracted from oil plants. 
Preferably, it is obtained from plants belonging to the camellia family, 
and most preferably is obtained from leaves and seeds of Camellia plants. 
The process used in the present invention for the production of 
triterpenoid saponin is described as follows. 
Seed cake remaining after oil extraction is grounded, then soaked in 
alcohol and other organic reagents. The organic extract is filtered, 
condensed and dried to obtain triterpenoid saponin powder. Temperature 
ranges from 20 to 50.degree. C., and preferably from 30 to 40.degree. C. 
Concentration of the organic range rangents from 60 to 90%. The drying 
method can be vacunm-drying or sprap-drying. 
Saponin obtained according to this invention can be used directly as feed 
additive or health care agent. It can also be used in combination with 
trace elements. 
Dosage and application methods of this saponin preparation are similar to 
those used for conventional feed additives and health care agents. Its 
content in feed can be in the range of 50-1500 ppm, and is preferably in 
the range of 250-750 ppm. 
This invention offers a bioactive ingredient from natural Camellia seed 
cake to be used as a feed additive. It can replace antibiotics in 
conventional feed completely (thus avoiding antibiotic residues in animal 
products), produce high-quality animal products, reduce environment 
pollution and increase animal production performances. It can also be used 
as a nutritive health care agent. Therefore, low valued oil-seed meal can 
be used effectively and have a better social and economic benefit. 
The following examples are used to illustrate the invention in more detail, 
but they are not intended to limit the scope of the present invention in 
any way.

EXAMPLE 1 
Preparation of bioactive feed additive A 
Eight thousand grams of Camellia olefera seed cake were extracted three 
times with 83 percent alcohol (v/v). All the organic extracts were 
combined and vacuum-dried. Eight Hundred grams of extracted powder were 
obtained, which was used directly as a feed additive. 
EXAMPLE 2 
Preparation of bioactive feed additive B 
Twenty five grams of zinc sulfate of feed grade and 25 grams of manganese 
sulfate of feed grade were added to 1000 grams of the feed additive A 
obtained in Example 1. After mixing evenly, feed additive B was then 
obtained. 
EXAMPLE 3 
Preparation of bioactive feed additive C 
One Hundred grams of zinc sulfate of feed grade, 100 grams of manganese 
sulfate of feed grade and 50 grams of vitamin C were added to 1000 grams 
of the above mentioned additive A. After mixing evenly, feed additive C 
was then obtained. 
EXAMPLE 4 
Improvement of survival rate of livestock 
From July 1992 to June, 1994, 3108 one-day old chicken (4 batches), 320 
eleven-day old piglets (8 batches) and 600 growing chicken for egg purpose 
were allocated into control and test groups to determine survival rates. 
Feed used in control groups were AMV broiler complete feed, PCS pig 
complete feed and Wanghai brand complete feed for chicken layers. The 
formulations of the three feed compositions were as follows: 
______________________________________ 
Formulation of PCS Complete Feed for Pig 
Ingredients piglet period 
growing period 
______________________________________ 
corn (%) 51.7 48.2 
wheat bran (%) 16.00 15.0 
middlings (%) 10.0 20.0 
fish meal (%) 6.0 4.0 
limestone (%) 1.0 1.0 
CaHPO.sub.3 (%) 0.5 0.5 
NaCl (%) 0.3 0.3 
additives (%) 1.0 1.0 
Olaquindox (ppm) 80.0 60.0 
______________________________________ 
AMV Complete feed for broiler 
Ingredients 0-21 days 22-49 days 
______________________________________ 
corn (%) 57 67 
soybean meal (%) 30 23 
middilings (%) 5 -- 
concentrate 1 (%) 5 -- 
concentrate 2 (%) -- 5 
fishmeal (%) 3.0 2.0 
yeast (%) -- 3.0 
colistin sulfate (ppm) 6.0 6.0 
bacitracin zinc (ppm) 30.0 30.0 
coccidiostatics (ppm) -- 125 (22-42 days) 
______________________________________ 
Wanghai brand feed for growing layers 
______________________________________ 
corn % 60 
wheat bran (%) 10.0 
soybean meal (%) 17.0 
fishmeal (%) 9.0 
bone meal (%) 2.0 
additive (%) 2.0 
terramycin (ppm) 100 
______________________________________ 
In test groups, 500 ppm of bioactive additive obtained from practical 
Example 1 was used to replace all the antibiotics in the control diets. 
The survival rates after 49, 120 and 21 days of experiments were estimated 
for broilers, piglets and growing layers respectively. The results are 
listed in Table 1. 
TABLE 1 
______________________________________ 
Survival rates of livestock 
growing 
broiler layers piglet 
survival survival survival 
group tested rate (%) tested rate (%) tested rate(%) 
______________________________________ 
saponin 
1845 95.30 300 88.00** 
160 100.00 
antibiotics 1263 94.4 300 77.00 160 98.13 
______________________________________ 
**P &lt; 0.01 
Results indicated that Sasanqua saponin could be used to replace 
antibiotics in the diets. The survival rates were increased by 0.95%, 
14.29% (P&lt;0.01) and 1.91% respectively for broilers, growing layers and 
piglets. 
EXAMPLE 5 
Effect of increasing live weight gain of livestock 
Under the same experimental designs as in example 4, the effects of the 
bioactive agents obtained by this invention on body weight gain were 
tested at day 49 (Table 2). The results were showed in table 2 below. 
TABLE 2 
______________________________________ 
Body weight gain of livestock 
broiler piglet 
net gain ADG net gain 
ADG 
group tested (g/each) (g/day) tested (kg/each) (g/day) 
______________________________________ 
saponin 
1845 2019.8 41.2 160 25.17 508.27 
antibiotics 1263 1971.2 40.2 160 22.92 463.60 
______________________________________ 
In broilers, net gain increased by 48.6 grams/bird and ADG increased by 
2.49%. In piglet, net gain increased by 2.25 kg/animal and ADG increased 
by 9.64%. 
EXAMPLE 6 
Effect of increasing feed efficiency 
Under the same experimental conditions as described in Example 4, the 
effects of the feed additive of the present invention on feed efficiency 
were tested. The results were showed in Table 3. 
TABLE 3 
______________________________________ 
Feed efficiency 
broiler piglet 
group tested feed:gain tested 
feed:gain 
______________________________________ 
saponin 1845 2.06:1** 160 2.42:1 
antibiotics 1263 2.26:1 160 2.54:1 
______________________________________ 
**: p &lt; 0.05 
Results suggest that replacement of antibiotics with bioactive saponin 
decreased ratio of feed-to-gain by 9.71% (P&lt;0.05) and 4.72% respectively 
for broilers and piglets. 
EXAMPLE 7 
Effect of improving meat nutrient contents 
Under the same experimental design as in Example 4, fat and amino acid 
profiles in chicken at day 49 were determined. Results suggest that 
saponin could improve chicken nutrient quality, especially threonine 
content by 8.38% (P&lt;0.05). 
EXAMPLE 8 
Effect of improving processing value of livestock products 
Under the same experimental design as in Example 4, some processing indices 
at day 49 were tested. Saponin replacement increased dressing percentage 
and total meat pigment by 1.93% and 7.48% respectively in broilers. Meat 
water loss percentage and pH decreased by 1.37% and 2.50% respectively. 
Results indicate that saponin addition is beneficial to meat processing 
and storage. 
EXAMPLE 9 
Effect of reducing heavy metals in meat 
Under the same experimental design as in Example 4, contents of heavy metal 
elements in meat were determined with an atomic absorption spectrometer at 
day 49. Results indicated that saponin reduced Cd and Pb contents in meat 
by 94.41 and 38.28% in broilers. 
EXAMPLE 10 
Effect of antioxidation 
To conventional feed, 750 ppm bioactive agent obtained in Example 1 was 
added. After incubation for 49 days at 40.degree. C., acidity of the feed 
was determined by KOH titration method, peroxidation value was determined 
by sodium thiosulfate method, and the content of Vitamin A was determined 
by HPLC. Results suggested that acidity and peroxidation value were 
reduced by 38.79% and 21.28% (P&lt;0.05) respectively, and saponin had 
protection over fat and Vitamin A. 
EXAMPLE 11 
Effect of increasing immune function and antiviruses 
Saponin obtained from Example 1 was added into broiler feed at a dosage of 
750 ppm. After IBD viruses were injected into chickens. at day 28, blood 
and spleen samples were taken at day 34. As a result, immunoglobin; T 
lymphocytes transformation rate, interleukin 2(IL-2) and erythrocyte C3b 
receptors were increased by 11.56% (P&lt;0.01), 54.09% (P&lt;0.05), 52.66% 
(P&lt;0.05) and 21.71% (P&lt;0.05) respectively. This suggests that saponin 
could improve immune function and has an anti-viral effect. 
EXAMPLE 12 
Effect of free radical scavenge and antimutation 
Light emision analysis results showed that saponin obtained from Example 1 
cleared about 94.40% of superoxide anion radical (O.sub.2.sup.-) (P&lt;0.01) 
and 78.19%(P&lt;0.01) hydroxyl radical (OH.sup.-). In adult cocks and hens, 
ethyl methanesulfonate (EMS) was used at the dosage of 82 mg/kg and 80 
mg/kg respectively via intramuscular injection for three days. Sasanqua 
saponin addition at 750 ppm decreased cock sperm deformation rate by 
58.96% (P&lt;0.01), increased hen ovulation rate by 28.64%, and increased egg 
fertility by 75.54 (P&lt;0.01). These results suggested that the extracted 
bioactive additive was effective in protecting cells against deformation 
induced by alkylating agents. 
EXAMPLE 13 
Effect of anti-bacteria 
Seven hundred fifty ppm extracted bioactive agent was added into a culture 
dish to evaluate its antibacterial effect. Results indicate that 
suppressing concentrations for E. Coli and salmonella were 1.25-0.5 mg/mL 
and 0.1563-0.0390 mg/mL respectively. 
EXAMPLE 14 
Effect of increasing hormone, protein and enzyme levels 
Under the same experimental design as in Example 4, venous blood samples 
were taken at day 49. Results showed that saponin addition increased serum 
testosterone level by 25.61% (P&lt;0.05), serum total protein content by 
11.08%, alkaline phosphatase activity by 19.023%, amylase activity by 
16.79% and total proteolytic enzyme activity by 49.37%. 
EXAMPLE 15 
Effect on growth of aquatics 
Addition of 10 ppm Sasanqua saponin in turtle, prawn and eel diets improved 
their survival rates and growth rates. 
EXAMPLE 16 
Safety test of Sasanquasaponin 
Tea saponin obtained from Example 1 was added at 5000 ppm in the diet of 
rats. After 30 days of feeding, no abnormal signs were observed in rat 
somatic cells and reproductive cells. Test results indicated that tea 
saponin is safe as a feed additive. 
It will be appreciated that it is not intended to limit the invention to 
the above examples only, many variations thereto and modifications thereof 
being possible to one skilled in the art without departing from its scope, 
which is defined by the appended claims.