Review of Traditional Chinese Herbs Used in the Clinical Treatment of Canine Parvovirus Infection

Chinese medicinal herbs that have appeared useful for prevention and treatment of viral diseases and in particular, canine viruses, have attracted research attention in recent years. Canine parvovirus infection which can be associated with high morbidity and mortality continues to cause disease outbreaks due to lack of efficacy of preventative procedures and immunity failure of susceptible animals. Increasingly researchers have been evaluating herbs ranging from traditional formulas to self-made prescriptions that have anti-viral activity to improve treatment and control of canine parvovirus infection. In this review, Chinese herbal medicines that appear to provide both effective treatment and a complimentary adjunct to traditional Western methods of treatment are presented

Traditional Chinese medicine (TCM) which originated in ancient China has evolved over thousands of years.The Yellow Emperor's Inner Canon, the oldest recorded work of Chinese medical theory, was compiled around the first century BCE and was based on shorter texts from different medical lineages. 1,2 ased on these early principles, TCM practitioners use various forms of herbal medicine, acupuncture, massage (Tui-na), exercise (Qi-gong) and dietary therapy to treat a wide range of disease syndromes. 3,4 lthough TCM has a long history and has been used for thousands of years, it was not widely used or recognized by western countries until approximately 20 years ago. 5,6 t is gradually gaining acceptance as some TCM methods and herbs to prevent or treat disease demonstrate efficacy in both clinical and mechanistic research. 7,8  1970, Binn and coworkers first reported the isolation of a parvovirus from asymptomatic dogs. 9,10 2][13][14][15] Since then CPV-2 has continued to genetically evolve, with CPV 2a, 2b and 2c gradually replacing earlier strains. 16- 18Since 1979, nearly 108,000 articles, papers, numerous text chapters and monographs have been published on the subject of canine parvovirus.At present, canine parvovirus (CPV) infection is primarily caused by CPV-2 with clinical signs including severe vomiting, leukopenia, myocarditis and hemorrhagic gastroenteritis.Its infection rate is as high as 100%, while the case fatality ratio ranges from 10%-50%. 19,20 7][28] In this review, Chinese herbal medicines that appear to provide both effective treatment and a complimentary adjunct to traditional Western methods of treatment will be presented.It is hoped this review will contribute to an understanding of Chinese herbal medicines as an effective treatment for CPV and provide useful information for the development of more effective antiviral drugs.
copyright © 2016 by AJTCVM All Rights Reserved

CHINESE HERBAL FORMULAS WITH ANTI-CPV ACTIVITY
Traditional Chinese veterinary herbal formulas are a combination of compatible herbs in fixed dosages guided by TCM principles, and are usually derived from classical or well-known Chinese textbooks of medicine (e.g., Yuan Heng Liao Ma Ji, the first traditional Chinese veterinary medicine book). 29Currently, several traditional Chinese herbal formulas have been found to have a potentially beneficial effect for treating CPV.A brief outline of the antiviral pharmacology of the most commonly used Chinese herbal formulas is discussed below and summarized in Table 1.

Shao Yao Tang
This formula combines herbs with known antibacterial and antiviral properties and acts to drain Heat and Damp.The source of this herbal formula is Suo Wen Bing Ji Qi Yi Bao Ming Ji, written by Liu Yuan Su in the Song and Jin periods. 32It is a mixture of ten herbs: Shao Yao (Paeonia), Gan Cao (Glycyrrhiza), Huang Lian (Coptis), Huang Qin (Scutellaria), Mu Xiang (Saussurea), Bing Lang (Areca), Dang Gui (Angelica), Guan Gui (Cinnamomum) and Da Huang (Rheum).This formula combines herbs that resolve Phlegm, tonify the Spleen, and resolve Stagnation (Qi Stagnation and/or Blood Stagnation secondary to a toxin such as Heat). 30

Prescription 1:
This prescription created by the sixty-sixth Regiment of the Fourth Agricultural Division's Veterinary Station located in Xinjiang province and Boxing County's Animal Husbandry Bureau located in Shandong province contains Huang Lian (Coptis) 15 g, Huang Qin (Scutellaria) 15g, Ge Gen (Pueraria) 8g, Fu Ling (Poria) 10g, Che Qian Zi (Plantago) 6g, Di Yu (Sanguisorba) 8g, Hou Pu (Magnolia) 8g, Huo Xiang (Pogostemon) 6g, Zi Jin (Corydalis) 5g, Huang Qi (Astragalus) 12g, and Gan Cao (Glycyrrhiza) 8g.According to Ge Gen Huang Qin Huang Lian Tang's research, the cure rate of canine parvovirus disease with this formula approaches 96%. 33If the affected animal was treated early and the disease syndrome was mild, the cure rate among puppies 3-12 weeks old and adults extending in age to more than 5 years old was 100%.In contrast, treatment failure occurred when a dog presented with a dry and cracked nose, oral ulcers, no appetite, poor anal tone, and body temperature less than 102 degrees F°. 33 In particular, there was high mortality in dogs with severe hematochezia and vomiting; most likely associated with poor absorption of the active ingredient of the formula due to vomiting.

Prescription 2:
The Shenqin compound consisting of Ku Shen (Sophora), Ban Lan Gen (Isatis), Jin Yin Hua (Lonicera), Lian Qiao (Forsythia), Huang Qi (Astragalus), Bai Zhu (Atractylodes), and Dan Shen (Salvia) was developed by the Animal Medicine Department of Southwest University (Chongqing, China).The formula is based on the Eight Principles theory and is designed to treat affected animals by moving Blood, clearing Heat Toxin, and removing Damp.Liu Junwei et al. reported that Shenqin compound could up-regulate the expression of Bcl-2 mRNA (antiapoptotic protein expression) and down-regulate the Bax mRNA (apoptotic activator protein)..

Prescription 3:
Compound Kuqin, based on Eight Principles and Zang Fu patterns, contains Ku Shen (Sophora), Huang Qin (Scutellaria), Huang Qi (Astragalus), Jin Yin Hua (Lonicera), Guang Huo Xiang (Pogostemon), Bai Tou Weng (Pulsatilla), Zhi Zi (Gardenia) and Bai Zhu (Atractylodes).Formula characteristics include Heatclearing and treating diarrhea (astringent qualities of Sophora and Scutellariae); and tonifying the Spleen by Astragalus and Rhizoma Atractylodes. 36,37 ompound Kuqin demonstrated a protective function against canine parvovirus in dogs receiving injections (0.5mL/kg body weight, three times per a day for 3 consecutive days) of the compound.Zhu Zhaorong reported survival rate reached 90% in dogs dosed with Compound Kuqin and then injected with CPV and recovery of the Compound Kuqin treatment group when clinically sick which reached 70%.8. copyright and Huang Qi (Astragalus). 39The IC50 against CPV was 1:587.5.In addition, the formula had an inhibitory effect against Escherichia coli, salmonella and staphylococcus aureus with 6.25 mg, 25 mg and 12.5 mg, respectively, in MIC assays.It has yet to be used in clinical cases but shows promise.

SINGLE CHINESE HERBS WITH ANTI-CPV ACTIVITY Lysimachia
][42] Research at Shaoxing People's Hospital (in Shaoxing, Zhejiang Province) demonstrated that Ma Ti Xiang could suppress peristalsis and shorten the time of diarrhea secondary to severe enteritis. 43It was also reported that Ma Ti Xiang was curative for CPV disease with resolution of intestinal mucosal lesions. 44

CHINESE HERBAL EXTRACTIONS WITH ANTI-CPV ACTIVITY
In recent years, active research centered on producing anti-viral medications from biologically active components of Chinese herbal medicines has increased.The most popular extractions are polysaccharides, alkaloids and flavonoids, which are being evaluated for their anti-viral activity.

Polysaccharides
Polysaccharides are polymeric carbohydrate molecules composed of long chains of monosaccharide units bound together by glycosidic linkages.It is the form in which most natural carbohydrates occur and hydrolysis will break them into constituent monosaccharides or oligosaccharides.Examples include starch, glycogen, cellulose, and chitin.Current research has demonstrated that polysaccharides have anti-viral and anti-cancer effects along with immunomodulation, which stimulates protective immunity.
Feng et al. reported that Astragalus polysaccharides (APS), Rhizoma Atractylodes polysaccharides (RAPS), root of Baikal skullcap polysaccharide (BSPS) and Flos lonicerae polysaccharide (FLPS) are biologically active polysaccharide ingredients of compound Kuqin..Additional work reported by Gou Peng, demonstrated the alcohol extract precipitation of Cass nomame (sieb) honda, which is a polysaccharide, resisted canine parvovirus infection in-vitro with an inhibition rate of 55.24%. 39The inhibition rate of its composite consisting of Cass nomame (sieb) honda, Astragalus and Coptis was 63.04% and the composite could inhibit drug-resistant strains of Escherichia coli and S cholera along with enhancement of effective lymphocyte transformation. 39n a review of anti-viral activities of the pinon shell polysaccharide (PSP), which is extracted from Korean pine in China, it was noted that this polysaccharide displayed a distinct antiviral effect at different concentrations on two viruses, one of which was CPV.At the lowest concentration (31.2 mg/liter), cell survival rates were above 49.08% for all three methods used (pretreatment, pretreatment/ posttreatment, posttreatment). 48

Flavonoids
Flavonoids are widely distributed in plants, fulfilling many functions.They are the most important plant pigments for flower coloration as well as producing yellow or red/blue pigmentation in petals designed to attract pollinators. 49,50 n higher plants, flavonoids are involved in UV filtration, symbiotic nitrogen fixation and floral pigmentation.Their dietary intake is quite high compared to other dietary antioxidants like vitamins C and E. 51,52 Studies evaluating flavonoid activity have been plentiful in recent years. 53Feng et al. demonstrated that the Flos Lonicerae flavone (FLF) from Compound Kuqin, at a dose of 15.625-7.813mg/Lcan inhibit CPV proliferation in F81 fetal cell lines. 47Carvalho et al evaluated the effect of the flavonoid, quercetin, on canine parvovirus replication in-vitro through a series of assays.Test 1 determined its viracidal activity, Test 2 looked at activity on the cell and test three, its effect (time assay) on steps of the viral replication cycle.Quercetin showed significant antiviral activity, with maximum values of 96.3% virus reduction in test 1, and 90% for tests 2 and 3.The most significant effects occurred in adhesion and viral penetration. 54.

Alkaloids
Alkaloids are a group of naturally occurring chemicals that are made by plants and are organic nitrogen containing bases.][57][58] Alkaloids from northern Nigeria plants with a history of use in both human and veterinary traditional medicine were tested for viracidal activity against poliovirus, astrovirus, herpes simplex viruses and parvovirus, using microtitre plate inhibition tests at a dose rate between 100 and 400 μg/100 μl. 59Wang et al. reported that the alkaloids from Corydalis were able to inhibit HIV-1 reverse transcriptase. 60ymerson et al. reported that N. tabacum cultivars (containing 81V9 alkaloid) derived from the upper leaves enhances the ability of serum antibodies to neutralize the infectivity of porcine parvovirus as demonstrated by a serum neutralization assay (1:2700-1:3900).This suggests the alkaloid (81V9) associated with the N. tabacum leaves may mimic VP2 (major protective surface antigen of porcine parvovirus) and has potential to improve the efficacy of a vaccine against this virus. 61

CONCLUSION
Based upon review of current scientific literature and upon prior knowledge and experience of the author, Chinese herbal medicines (including Chinese herbal formulas, single herbs and related biologically-active compounds) that appear effective and worthy of additional in-depth study in treating CPV were selected for presentation.Experimental results from both mechanistic and clinical studies demonstrate that there is mounting evidence that some Chinese herbal medicines (e.g.Yu Jin San, Shao Yao Tang and Compound Kuqin) can be an effective treatment for clinical cases of canine parvovirus.Further investigation in well-designed clinical trials will yield a better understanding of mechanisms, therapeutic effects, and the safety profile of Chinese herbal medicine used to treat canine parvovirus disease and will give clinicians another tool to combat this serious canine disease.

Table 1 :
© 2016 by AJTCVM All Rights Reserved Traditional Chinese herbal formulas with anti-CPV activities copyright © 2016 by AJTCVM All Rights Reserved In a survey on the antiviral effects of Cass nomame (sieb) Honda, it was superior to other anti-viral extracts applied to F81 cells with a 63.04% inhibition rate of its composite.Gou Peng et al. (Medical Laboratory Animal Center of PLA General Hospital) reported this formula was mainly composed of Shan Cha (Camellia japonica L.)