J. Vet. Med. A 39, 593-599 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931 - 184X

'Department of Animal Science and Veterinary Medicine, Zhejiang Agricultural University, Hangzhou, Zhejiang, P. R. China 2 Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, P. R. China 3 Institute of Nuclear Agricultural Science, Zhejiang Agricultural University, Hangzhou, Zhejiang, P. R . China

Influence of Medicinal Herbs on Phagocytosis by Bovine Neutrophils SONGHUA Hut, WEIMINGCAI', JUNANYE',ZHIJIANQ I A Nand ~ ZHIMINGSUN' Address of authors: Department of Veterinary Microbiology, College of Veterinary Medicine, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden

With 2 tables (Received for publication March 2, 1992)

Summary Twenty-six herbal preparations made from 24 medicinal herbs, categorized as antipyretics in Chinese materia medica, were tested in witro to determine their effects upon phagocytosis of 3ZP-labelled Staphylococcus aureus by neutrophils isolated from bovine blood and milk. The percentage of phagocytosis was determined after incubating (1 hour at 37°C) 1.25 x 107 neutrophils, 1 x lo8 32P-labelled S. aureus and 10 % skimmed milk with herbal solutions. Concentrations of herbal preparations tested were 100 YO,10 YO and 1 % (v/v). When compared with PBSS (0.01 M phosphate buffered saline solution) controls, most of the herbs at high concentrations inhibited phagocytosis while at lower concentrations phagocytosis was increased. All 26 herbal preparations significantly increased blood neutrophil activity at their proper concentrations. The most active herbs in promoting blood neutrophil phagocytosis were observed for Herba werbenae, Flos chtysanthemi, Flos lonicerae, Radix sophorae fivescentis, Herba houttuyniae, Radix isatidis, Herba patriniae, Berberini sxlfatis at the lowest concentration and for Folium hibisci at the high concentration with the increased percentage of more than 40.0 % in comparison with PBSS control. Of the 19 herbal preparations tested with milk neutrophils, 18 herbs greatly increased phagocytosis at suitable concentrations. The most active preparations in stimulating milk neutrophil functions included Folium hibisci, Flos chtysanthemi, Radix bupleuri, Radix stellariue, Herba houttuyniae, Herba senecionis scandentis, Caulis lonicerae and Flos lonicerae which increased phagocytosis by over 35.0 %.

Introduction Antibiotics have been utilized for the treatment of bovine mastitis for about 50 years. They are still a major therapeutic method and are widely used in dairy farms. However, the extensive use of antibiotics in the dairy industry has caused potential residual problems in milk and its by-products and may jeopardize public health. Therefore, non-antibiotic approaches, such as improving host specific immunological systems and increasing neutrophi1 phagocytosis, have become a topic of interest to many researchers. Medicinal herbs as a form of natural therapy have been employed in China for thousands of years. Some herbal preparations administered orally have been proven to be U.S.Copyright Clearance Center Code Statement:

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effective in the treatment of bovine mastitis (1, 2). The main ingredients of these preparations are the herbs categorized as antipyretics in TCVM (Traditional Chinese Veterinary Medicine). In witro tests showed that these herbs had antibacterial activities against mastitic pathogens such as Staphylococcus aureus and Streptococcus agakactiae (3). Other research indicated that some herbs significantly increased phagocytosis by mouse phagocytes (4, 5). Although much has been learned regarding the antibacterial activities of the herbs and their effects on phagocytosis by human or mouse phagocytes, the interaction between herbs, bacteria and bovine neutrophils has, to our knowledge, not been studied. The purpose of the study was to determine the effect of 26 herbal preparations on phagocytosis by bovine neutrophils and to explore the feasibility of developing a herbal intramammary injection for the treatment of bovine mastitis. Material and Methods Cattle Three lactating Holstein cows were used as source of neutrophils. Based upon bacteriological culturing of foremilk on blood esculin agar (beef extract 3 g, peptone 10 g, sodium chloride 5 g, esculin 1 g, agar 20 g and distilled water 1,000 ml), these cows were determined to be free of intramammary pathogens. Milk somatic cell count (SCC) was measured with a Coulter counter (Model ZM, Coulter Electronics LID, England) and the SCC from each quarter's foremilk sample was less than 200,000/mL Neutrophils For isolation of blood neutrophils, 600ml of blood was collected from each cow in a heparinized (40 IU/ml) flask by means of jugular venipuncture. Samples and reagents were kept on ice during the isolation procedure. Neutrophils were isolated as previously described (6) and adjusted to 2.5 x 107/ml. O n the average, 85 % of the cells recovered were neutrophils with 96 % viability. Mammary gland neutrophils were isolated from lactating cows 12 hours after intramammary infusion of 100ml sterile PBSS (0.01 M phosphate-buffered saline solution) containing 0.1 % oyster glycogen (Sigma Chemical Co. St.Louis, Mo, USA). After washing and massage of the udder, approximately 700 ml of milk was milked out from one quarter and then 600 ml of residual milk was collected from the quarter into a flask. The neutrophil isolation was as described (7) and the suspension was adjusted to a desired concentration using a procedure similar to that used for blood neutrophils. O n the average, 96 of the cells recovered were neutrophils, showing 97 YO viability. Herbal Drugs A total of 26 herbal preparations were prepared from 24 medicinal herbs belonging to the category of antipyretics. Four preparations were purchased from a drugstore: Injection isatidis (Peace Drug Manufacturer, Guangdong Province, China), corresponding to 0.5g of the dried Radix isatidis per ml; Injection Caulis lonicerae (Znjectio Antiparotiditis [trade name], Beijing No. 4 Drug Manufacturer, Beijing, China), corresponding to 0.5g of the dried Caulis lonicerae per ml; Injection Corydalis decumbentis (Yujiang Drug Manufaccurer, Jiarigxi Province, China), containing 0.5 mg alkaloid extracted from Corydalis decumbentis per ml and Injection Berberini sulfatis (Gaoyi Animal Drug Manufacturer, Shanghai, China), containing 1 .O % of Berberzni sulfatis. The preparations of Herba houttuyniae, Radix bupleuri, Folium artemisiae argyi and Oleum lonicerae were made by distillation with water (8). Each ml of the preparations were made from 2 g of the dried herbs. Fourteen preparations were made from the following 14 herbs: Herba oldenlandia, Herba violae, Rhizoma fagopyri cymosi, Herba portulacae, Radix sophorae flavescentis, FIos lonicerae, Herba ajugae, Herba verbenae, Herba lobeliae chinensis, Herba hyperici japonici, Herba patriniae, Herba senicionis scandentis, Herba taraxaci and Folium hibisci (8). Their final concentration was adjusted with distilled water to that corresponding to 2 g of the dried herb per ml. The remaining four preparations were as follows: Baicalin preparation. It was extracted with water from Radix scutellariae, containing 20 mg baicalin per ml (9). Andrographolide bisulfite preparation. Andrographolide was extracted from Herba andrographiris and purified. The purified product was then reacted with sodium bisulfite to form andrographolide bisulfite. Its concentration was 50 mg per ml (8).

595

Medicinal Herbs and Neutrophil Phagocytosis

Chrysuntherni preparation. It consisted of 80 mg of the dried extract made from Flos chryysunthemi indici (8). Oleum eucufypti preparation. It contained 0.25 YOof Oleum eucalypti distilled with water from Eucalyptus globulus labill (8). Each of 26 preparations was diluted with distilled water to three concentration levels: 100 %, 10 % and 1 % for use in the assays. Fhugocytosis In this procedure, tube A and tube B were prepared and the assay procedure was performed as previously described (lo), except that 7ml polypropylene centrifuge tubes were used to replace 25-mI Erlenmeyer flasks and the incubation mixture included 1ml of herbal preparation or PBSS,0.5 ml of 10 % skimmed milk and 0.5 ml3*P-labelled Stupbylococcus uureus (1 x 108) and 0.5 ml of the neutrophil suspension. To eliminate the quench effect caused by some colored herbal preparations, a set C was prepared for every herbal solution. This was made as follows: one ml of drug, 0.5 ml of 10 % skimmed milk and 0.5 ml of 32P-labelled S.aureus were added into tube C. This mixture was incubated at 3 7 ° C

Table 1. Influence of 26 herbal preparations on phagocytosis by blood neutrophils Herbal preparations

Botanical source

Concentrations (%) 100 10 1

Herbu verbenae Flos chvsanthemi indici Herba hyperici japonici Flos lonicerue Radix sophorae fivescentis Herba honttnyniae Folium hibisn' Radix isatidis Herba patrinae Berberini sufatis Caulis lonicerue Herba senecionis scundentis Andrographide bisnlphate Herba ajngae Radix bnplenri Radix scutellariae Herba oldenlandia Herba lobeliae radiantis Rhizoma corydalis decumbentis

Verbena officinalis L. (whole plant)

-85.5" -34.8'

Chrysanthemum indicnm L. (flower) Hypericnm juponicum Thunb. (whole plant) Lonicera japonica Thunb. (flower)

-85.6' NA -11.4'

Sophora fivescens Ait. (root) Honttuynia cordata Thunb. (whole plant)

-34.3" - 7.5' -23.6* -13.9' 42.1' 31.1" -13.6" - 2.6 -35.0' 1.9 15.3" 28.1' -75.6" 8.7"

Herba portnlacae Herba vwlae Herba tararaci Oleum argyi Olenm eucalypti Oleum lonicerue Rhizoma fagopp' Cymosi

Hibiscus mutabilis L. (leaf) Zsatis tinctoria L. (root) Patrina scabwsaefolia Fisch. (whole plant) Coptis chinensis Franch. (root tuber) Lonicera japonica Thunb. (stem) Senecio scandens Buch.-Ham. (whole plant)

NA

46.1'

- 2.8

44.8' 6.8 44.0' 30.3" 43.8' 42.8" 42.1" 21.2' 41.0" 40.9' 40.6" 39.7"

6.4' 37.5"

Andrographis paniculatu (Burm. f.) Nees (whole plant) Ajngae decumbens Thunb. (whole plant) Bnoleurum chinensis DC. (root) Scutellaria baicalensis Georgi (root) Oldenlandia diffusa willd. (whole plant)

-21.1" 24.6' 35.7' -89.4" 32.9' 32.9' -55.0" 21.4' 32.7' - 8.7" -26.6" 32.6' -51.0' - 7.6" 30.4*

Lobelia chinensis Thunb. (whole plant)

-77.1*

10.1" 30.4"

corydalis decumbens Thunb. (root tuber) Portulaca oleracea L. (whole plant) Viola yedoensis Makino (whole plant) Tarmacurn mongolicum Hand.-Man. (whole plant) Artemisit urgyi Led. et Vant. (leaf) Eucalyptus globnlns Labill. (leaf) Lonicera japonica Thunb. (flower)

-52.0' -82.2" -73.1" -60.0" -81.9" -65.0"

Fagopyrnm cymosum (Trev.) Meissn (root tuber)

-68.7" -21.9* 10.4'

28.7*

17.7" 8.0 -15.6" 21.9" 11.6" -17.3' -34.0" -35.8'

26.1' 23.7' 21.5' 15.8' 14.3* 11.7"

' P < 0.05, compared with PBSS control. Data are expressed as the percentage of change relative to PBSS control. NA - Not applicable.

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SONGHUAH u , WEIMINGCAI,JUNANYE, ZHIJIANQIANand ZHIMINGSUN

in a shaking water bath for 30 minutes. Then 3 ml of PBSS containing 5 IU lysostaphin (Sigma Chemical Co. St.Louis, Mo, USA) was added to it and incubated at 37°C for 60 minutes with agitation for the first 15 minutes. After that, the tube was centrifuged at 1,500 g for 30 minutes at 4 "C. O n e ml of the supernatant was added to 9 ml distilled water and counted in a scintillation spectrometer (Packard 1900 TR, Canberra Company, counting efficacy for P > 33 %). The percentage phagocytosis was determined by the equation: P= ( C - A ) / ( C - B ) X 100 All determinations were performed in duplicate. Statistical analysis A t test was used to compare the percentage means of phagocytosis and those from PBSS control. The significance level was set at P < 0.05 (11).

Results Generally, most of the drugs at the high concentration inhibited phagocytosis while at lower concentrations increased phagocytosis was observed (Table 1 and Table 2). Significant increases ( P < 0.05) in phagocytosis by blood neutrophils were found for all of the 26 herbal preparations at their proper concentrations (Table 1). Bevberine sulfatis and Herba hibisci greatly increased phagocytosis at all three concentration levels ( P < 0.05). With the decreased concentrations of drugs, the stimulatory effects of Rhizoma corydalis decumbentis and Folium hibisci on phagocytosis was reduced (Table 1).

Table 2. Influence of 19 herbal preparations o n phagocytosis by milk neutrophils Herbal preparations

Botanical source

Concentrations (%) 100

Folium hibisci Flos chrysanthemi indici Radix bupleuri Radix scutellariae Herba houttuyniae Herba senecionis scandentis Caulis lonicerae Flos lonicerae Radix isatidis Andrographide bisulphate Radix sophorae flavescentis Rhizoma fagopyri cymosi Herba verbenae Herba portulacae Herba violae Herba odenlandia Rhizoma corydalis decumbentis Herba ajugae Oleum eucalypti

Hibiscus mutalis L. (leaf)

10

1

40.5" -42.7" 26.2"

Chrysanthemum indicum L. (flower) Bupleurum chinensis DC. (root) Scutellaria baicalensis Georgi (root) Houttuynia cordata Thunb. (whole plant)

-44.0~- -23.12- 41.y -33.4:' 15.4" 40.3" 2.8 0.2 38.6'' -57.3" -32.5" 36.9"

Senecio scandens Buch.-Ham. (whole plant) Lonicera japonica Thunb. (stem) Lonicera japonica Thunb. (flower) Isatis tinaaria L. (root)

NA 5.1 -76.5" 16.5' -33.7 11.5 -31.82. -12.1:'

36.6" 36.5" 35.8" 34.1"

Andrographis paniculata (Burm. f.) Nees (whole plant) Sophora flavescens Ait. (root) Fagopyrum cymosum (Trev.) Meissn (root tuber) Verbena officialis L. (whole plant) Portulaca oleracea L. (whole plant) Viola yedoensis Makino (whole plant) Oldenlandia d;ff.sa willd. (whole plant)

-52.2" -26.9'' -56.4" -49.3'. -32.6-

Corydalis decumbens Thunb. (root tuber) Ajugae decumbens Thunb. (whole plant) Eucalyptus globulus Labill. (leaf)

1 5 . 7 26.2" 12.3'' -57.3'1 - 0.2 18.1" -49.6". -41.6 2.0

-34.5" 32.3" -48.2'' 32.2" -21.5" 28.9" -31.5" 27.8" -25.9" 27.3"

't P < 0.05, compared with PBSS control. Data are expressed as the percentage of change relative to PBSS control. NA - Not applicable.

Medicinal Herbs and Neutrophil Phagocytosis

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The most active herbs in promoting blood neutrophil phagocytosis were Herba verbenae, Flos chrysanthemi indici, Flos lonicerae, Radix sophorae jLvescentis, Herba houttuyniue, Radix isatidis, Herba patriniae, Berberini sulfatis at the lowest concentration and for Folium hibisci at the high concentration with an increased percentage of more than 40.0 YO compared with PBSS control (Table 1) Of the 19 herbal preparations (Table 2), 18 drugs, at their appropriate concentrations, significantly increased milk neutrophil functions. No effect on phagocytosis was observed at the lowest concentration for Oleum eucalypti, whereas at both high and intermediate concentrations, phagocytosis was reduced 49.6 and 41.6 % respectively. The most active preparations in stimulating milk neutrophil phagocytosis included Folium hibisci, Flos chrysantherni, Radix bupleuri, Radix scutellariae, Herba houttuyniue, Herba senecionis scandentis, Caulis lonicerae and Flos lonicerae. They increased phagocytosis by more than 35.0 %.

Discussion Antipyretic herbs are traditionally used in China to treat infectious diseases such as endometritis, pneumonia, enteritis and mastitis, etc. (12). How do they work pharmacologically on these diseases? Some reports explain that this is based only upon their bactericidal properties (13). However significant antibacterial action is difficult to explain due to the fact that most of the herbal drugs, after oral administration, are unable to reach a high concentration within the body to inhibit pathogenic microorganisms. For example, berberine, a pharmacologically active constituent extracted from Coptis chinensis France, was clinically effective in treating carbuncle, tonsillitis and bacterial toxemia (12). Its in vitro bactericidal concentration was 125 pg-20 mg/100 ml, while its peak blood concentration after per 0s administration was only 100 pg/IOO ml, which was much lower than that required for a bactericidal effect (14). Previous investigations demonstrated that a number of herbs used to treat infectious diseases could significantly increase phagocytosis by mouse macrophagocytes (5, 15). In our experiment, similar results were obtained for 26 herbal preparations using blood neutrophils. Increased bovine milk neutrophil function was also found for 18 of the 19 herbs tested. Our research demonstrated that most of the herbs had a dose-dependent effect on phagocytosis: enhancement at a lower concentration and suppression at a high concentration. Those results agreed with previous studies (16). This phenomenon conformed with the fact that the dosage of a herb in clinical use is often not so high as that needed for bacterial inhibition. All of this suggests that the therapeutic effects of medicinal herbs on infectious diseases may be chiefly attributed to their ability to improve the host defence mechanisms by increasing phagocytic activity. Many antibiotics were efficacious but significantly reduced neutrophil phagocytic and bactericidal activity (17, 18, 19,20). The herbs tested in this study were shown to stimulate neutrophil function. It is recommended that to minimize the anti-phagocytic effect in mastitis treatment, antibiotics and herbs could be used together. The degree of the effect of a herb on phagocytosis by blood neutrophils was different from that by milk neutrophils. For instance, Oleum eucalypti increased blood neutrophil phagocytosis by 14.3% at the low concentration, while the same concentration had no effect on phagocytosis by milk neutrophils. Further studies are required to explain this result. Because antibiotic treatment for bovine mastitis results in a residual problem in milk, many Chinese researchers have begun to study herbal medicine. Some oral preparations made from medicinal herbs have been shown to be effective (1,2), but they were usually inconvenient for extensive application. Herbal intrarnammary preparations such as An-ru (a solution made from Oleum Eucalyptz] and Sbuang-ding (a solution made from Herba

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SONGHUA Hu, WEIMINGCAI,JUNANYE, ZHIJIANQIANand ZHIMINGSUN

taraxuci and Herba violae, etc.) have been reported to be effective (21,22). But they caused irritation to the udder. Their quality was also difficult to control since they were made from crude herbal extracts whose active parts were unknown. As a result, no one herbal product is widely used in dairy farms. Further study is necessary in order to develop a herbal intramammary preparation for practical use. After observing their effect in activating phagocytosis in blood and milk, special attention should be paid to Flos chrysantherni indici, Foliurn hibisci, Herba houttuyniae and Flos lonicerae. They are the herbs most commonly used in treating bacterial diseases. In wiwo testing showed that the boiled solution of Herba chrysantherni indici, after intravenously injected into mice, greatly increased macrophagocytic activity and effectivelyprotected mice from infection by S. aureus (4). Clinically, this has been used in the treatment of chronic pelvic inflammation, parotitis, dysentery and dermatitis etc (12). Foliurn hibisci has been used, in external or internal application, in treating mastitis (23). Experimentally, extracts either from Herba houttuyniae or from Flos lonicerae had an anti-inflammatory action and stimulatory activity on phagocytosis. Their clinical applications were reported effective for the treatment of pneumonia, bronchitis, rhinitis and uterine inflammation (12). Because of their ability to increase phagocytosis by neutrophils both from blood and from milk, and their effectiveness in the treatment of infectious diseases, the most active herbs in activating phagocytosis such as Flos chrysantherni indici, Foliurn hibisci, Herba houttuyniae and Flos lonicerae should be further studied so as to develop a non-antibiotic intramammary method to treat bovine mastitis. Acknowledgements We acknowledge financial support from the International Foundation for Science, Grev Turgatan 19, S-114 38 Stockholm, Sweden, and also thank Mr. H. YANG,Mr. H . R. LIU, Ms. X. Y. SHAOand Ms. A. F. D u for their technical assistance and to Dr. K. WATERSfor her careful proof reading.

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11. YANG,S.Q. et al., 1986: Health Statistics, 2nd ed. People’s Medical Publishing Press, Beijing, China. pp. 223-265 (in Chinese). 12. YAN,R. Q., 1982: Medicines for anti-pathogenic microorganisms. In: Wu, B.J. et al., Pharmacol-

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1055- 1061. 20. NICKERSON, S. C., M. J. PAAPE,R. J. HARMAON, and G. ZIV, 1986: Mammary leukocyte response to drug therapy. J. Dairy Sci. 69, 1733-1742. 21. Gu, X.G. P.Z. SHI, and F.Z. ZHOU, 1990: Studies on Injection “An-ru” for the treatment of bovine mastitis. Proceedings of the South China Association of Traditional Chinese Veterinary Medicine 9th Symposium. Jiangxi Province, China. pp. 30-31 (in Chinese). 22. LI, D. Y., Y. S. REN, and J. X. Wu et al., 1985: Injection “Shuang-Ding”for treatment of bovine mastitis. Chn. J. Vet. Med. 11, 47-49 (in Chinese). 23. CHEN,Y. C., and S. W. XIA, 1988: Folium Hibisici for treatment of acute inflammation of the skin in animals. J. Tradi. Chn. Vet. Med. (Lanzhou) 1, 39-40 (in Chinese).