Toxic effects of BuGuZhi and feasibility analysis of ways to synergistic and attenuated

Qian Cui,Yu-Hui Ma,Xiao-Ming Jin,Jing-Jing Liu,Yan-Hong Zhang,Guo-wei Zhang*

1College of Chinese Medicine,Hebei University,Baoding,Hebei province,China.

Background

BuGuZhi(Psoraleae Fructus)is a dry and ripe peel product of the leguminous plant.After ripening in autumn,the harvested fruit is dried in the sun that it is BuGuZhi.Its shape like a kidney,and the taste is spicy,bitter,warm.What is more,it belongs to the kidney and the spleen.Because of the shape and taste,it has the functions of warming the kidney and helping the yang,calming the breath,warming the kidney,stopping the diarrhea,and topical treatment of vitiligo,alopecia areata[1].The chemical components contained in BuGuZhi are complex,including Coumarins,which is mainly comprised by psoralen and is opsoralen,Flavonoids,which mainly contains Corylifolin,Corylifolinin,etc.and Monoterpene phenols with bakuchiol as the main component."Lei's treatise on preparing drugs"is the earliest ancient documentrecording thetoxicity ofBuGuZhi.The original text:"Where,the nature is very dry and poisonous,after soaking in wine for one night,and it is take-n out,it is soaked in the east stream for three days.However,steaming,from Si to Shen,out and dried day by day.”With the improvementofmodern medical,BuGuZhi extract is used in modern medical treatment,playing a critical role of antibacterial,anti-viral and anti-cancer.What’s more,BuGuZhi is widely used in vitiligo and other skin diseases because of its photosensitive components,which can eliminate the expression of vitiligo-related antibodies and block the destruction of melanocytes Cai Yu et al found that psoralen is cytotoxic to HL60 cells,which can induce tumor cell apoptosis,and is used in the adjuvant treatment of cancer[2].As an anti-cancer sensitizer for Chinese Medicine,it provides a new entry point for the reversal of cancer resistance.Meanwhile,some studies also have found that bakuchiol also has a certain effect on osteoporosis[3].As BuGuZhi has been widely used in clinical practice,the incidence of adverse reaction events has also increased.Therefore,the rational and safe use of traditional Chinese medicine is imminent.

Hepatotoxicity

Report on clinical cases of liver toxicity

Hepatotoxicity is the major side effect of BuGuZhi.More and more clinical reports have shown there are great number of cases of liver damage after taking BuGuZhi alone or in combination with other drugs.Naw SW et al reported a menopausal woman got cholestatic liver injury after taking BuGuZhi 10 times as more as the clinically usual does,suggesting the risk of hepatotoxicity in taking large doesmof BuGuZhi[4].Li Yingjun et al reported an elderly male who took medicated dietcontaining BuGuZhi(dose of 9 g)to treat senile urinary incontinence[5].Fifteen days later,he developed fever,nausea,chills and other symptoms.During the hospital admission,biliary tract diseases,viral hepatitis and other organic diseases were excluded.Because of abnormal elevation of biochemical indicators, he was diagnosed as drug-induced liver injury.Ma Rui et al reported a case of fever,nausea and other symptoms after 13 days of oral administration of compound granules containing BuGuZhi,and as it did in the case of a patient after abortion.After admission,biochemical tests showed:ALT,898 U/L;AST,421 U/L;GGT,323 U/L(normal liver function before abortion);the analysis of compound granules caused by liver damage is BuGuZhi,whose daily oral dose is 20 g.Considering that the dose of BuGuZhi exceeds the usual,the patient's constitution is weak after abortion,and large doses of BuGuZhi should be avoided for a long time[6].

Animal experimental study on hepatotoxicity

Study on hepatotoxicity induced by bakuchiol Li ZJ et al studied miceafter intragastric administration of bakuchiol for 6 weeks,and the results showed that the mice in high dose group(262.5 mg/kg)are weight loss and loss of appetite[7].Long-term using large doses of bakuchiolcausesliverdamagein mice,and bile salt-export pump(BSEP)is reduced,which may cause cholestatic liver poisoning.However,no significant hepatotoxicity was observed in the low-dose group(52.5 mg/kg).Bi YN et al used hypodermic injection of bakuchiol 50 mg/kg and estradiol 10 mg/kg to mice for 14 days[8].The results showed that estradiol is harmful to the liver,but the harm of the bakuchiol group is not obvious,the reason may be related to dose.

Study on hepatotoxicity induced by psoralen and isopsoralen Wang Anhong et al.using psoralen carried out experiments,and found that high dose(250 μmol/L)of psoralen had a significant inhibitory effect on BSEP,whose level will affect sodium taurocholate cotransporting polypeptide(NTCP)and increase its level,then increase the content of bile acids in liver cells,causing cholestatic liver damage[9].Song Lei et al.administered mice with isopsoralen 80 mg/kg and 40 mg/kg for 9 days and observed the liver injury[10].The blood biochemical examination showed that ALT and AST were significantly increased in the 80mg/kg group.Pathological findings showed that some hepatocytes in group 40mg/kg were swollen and denatured.Necrosis in the liver area of the 80 mg/kg group confirmed that isopsoralen can cause liver damage.Bai Ruyu et al.applied psoralen,isopsoralen and a mixture of the two to the mice in a large dose,and after observing for 14 days,they concluded that drug toxicity would occur to mice in this way[11].

Study on hepatotoxicity of BuGuZhi ethanol extract Wang J et al.administered SD rats with ethanol extract of BuGuZhi,the experiment was divided into three groups of different doses[12].After 28 days of gavage,the analysis showed that ethanol extract of BuGuZhi could cause cholestasis liver damage.The damage of female rats was extraordinarily obvious.When mice were administered with ethanol extract of BuGuZhi,the acute tests resulted in 70%mortality.Subacute experiments could make mice attacked by liver toxicity[13].

Study on hepatotoxicity of BuGuZhi aqueous extract There already has been some studies show that by applying BuGuZhi aqueous extracts to mice for acute experiments and subacute experiments,all of the mice died within 30 minutes in acute trails,and the mice showed a certain degree of hepatotoxicity in subacute trials in which the BuGuZhi aqueous extract was given 80 times as much as the clinical human dose[14].

Comparison of hepatotoxicity between aqueous extract and ethanolextract of BuGuZhi Chen Ying et al.carried out an experimental research with zebrafish by using BuGuZhi aqueous extract(main ingredients are psoralen, isopsoralen) and ethanol extract (main ingredient is bakuchiol)[15].The results showed that both the aqueous extract and ethanol extract could make the viscus of zebrafish poisoned,the result showed that both the high doses of psoralen and bakuchiol may cause hepatotoxicity as well,but the hepatotoxic effect of bakuchiol was stronger.The mechanism of hepatotoxicity was that bakuchiol and psoralen made BSEP decreased mainly by interfering with it to cause bile cracking liver damage.However,when the dose was reduced,the hepatotoxic component in BuGuZhi did not cause liver damage in rats,and BSEP was not significantly reduced.The hepatotoxicity of BuGuZhi is directly related to the used time and the administered dose.

Nephrotoxicity

Zhang Yushun et al.administered mice of bakuchiol with different doses and different number of days,the results showed that high dose(0.5 g/kg,25 times the usual adult dose),long time(21 days)group of mic-e appeared more serious kidney damage which is irreversible in a short time[16].Jiang Fang et al.suggested that bakuchiol has a destructive impact on kidney(HK-2 cytotoxicity)[17],and the cytotoxic effect of bakuchiol-l on HK-2 was significantly visibly reduced afteradding ratliver homogenate S9,the mechanism of which is the CYP enzyme converts bakuchiol to a non-toxic or low-toxic component[18-19].However,a larger dose(40umol/L)of bakuchiol is still renal cytotoxic even if activated by s9 metabolism.HK-2 cells called human renal tubular epithelial cells belongs to proximal tubule cell of normal kidneys.The toxic effect of bakuchiol on HK-2 cells is an important way to cause kidney injury.

Cytotoxicity

BuGuZhi has cytotoxicity and most studies focus on liver cytotoxicity.Ji Yubin et al.used Bavachinin to act on HepaRG cells and found that it can cause apoptosis[20].And as dosage increasing,the number of apoptosis related protein caspase-3,Bax,Bcl-2 increased as well.Wang S et al.study the mechanism of Bavachin on liver injury with HepaRG cells and found Bavachin could damage HepaRG cells through ROS,JNK and P38 signaling pathways[21].Some studies reported that Bavachin induced apoptosis of HepaRG cells should be related to mitochondrialdamage,the structure of mitochondria changes in the early stage of injury possession,mitochondrial membrane potential decreased significantly,which caused liver cells injury[22-23].At the same time,BuGuZhi can cause apoptosis of HpeG2 cells by inducing endoplasmic reticulum stress and mitochondrial function injury[24].Zhou Kun et al.found that psoralidine induced apoptosis of HepG2 cells,due to it acts on the endoplasmic reticulum and stimulates the release of Ca2+,Promoting mitochondrial PT pore to open[25].

Phototoxicity

The photochemical reaction initiated by the photosensitizer is called photoreaction reaction,including a phototoxic reaction and a photoallergic reaction.After the skin contact with the substance containing the furocoumarin and is exposed to ultraviolet rays at the same time,the sensitivity of the skin to ultraviolet rays is enhanced,which then cause an allergic reaction.BuGuZhicontainsphototoxicity substances,mainly composed of psoralen and methoxy psoralen.Among them,8-methoxy psoralen(8-MOP)is an important component of phototoxicity[26],it is mainly distributed in the skin[27].The side effects after touch were erythema,itching,and pigmentation spotting on the prognosis.At the same time,psoralen combined with ultraviolet long wave in the treatment of vitiligo has the risk of inducing squamous cell carcinoma[28].It has been reported that workers in the Chinese pharmaceutical factory have been touched to Chinese herbal compound containing BuGuZhi for a long time[29].And the patient developed erythema flaky and swelling rash after exposure to sunlight.So,bare-handed contact to water extracts containing large doses of BuGuZhi should be avoided in the presence of ultraviolet radiation.

When patients with vitiligo need to applicate the PUVA therapy for a long time,doctors should closely observe whether the patient suffer from the skin erythema and other allergies.Therefore doctors are suggested to follow up regularly.

Reproductive toxicity

Diawara M et al found that long term feeding mice with BuGuZhi at the Clinical equivalent dose can cause weight loss of the uterus,decreased ovarian,corpus luteum function and insufficient estrogen secretion [32].Reproductive toxicity is only found in animal experiments,and no such reports have appeared in the clinic.However,Tracing back to the ancient literature"Kai Bao Ben Cao"records:BuGuZhi treat"abortion of women's blood and gas".The injury of BuGuZhi to the uterus needs further scientific research.

Adverse reactions of BuGuZhi injection

Clinical cases have reported that the symptoms of nausea,vomiting,dizzinessand headache occurred in the treatment of vitiligo with BuGuZhi injection,and the symptoms gradually alleviated after stopping BuGuZhi injection[33].In recent years,the adverse reactions of injection are increasing,the reason of which might be that the impurities such as tannic acid and protein cannot be completely separated due to the lagging development of purification technology and unreasonable addition of excipients.

Side effects of blurred vision

Blurred vision resulted from taking 6 months of Chinese medicine containing BuGuZhi.After withdrawal for one week,the symptoms were relieved.According to the literature,it may be due to methoxy psoralen was absorbed into blood,which can cause retinal toxicity[32].

Research on processing technology in ways of synergistic and attenuated

The processing technology ofBuGuZhiwasfirst recorded in the"Processing Theory of Leigong".After generations of physicians continuously improved it on the basis of Leigong Processing,it mainly involved the methods of processing with wine,stir-frying,processing with salt-water,processing with urine of boys under twelve.Nowadays,BuGuZhi is usually processed by the Chinese Pharmacopoeia.In most places,the method of processing BuGuZhi is usually stri-frying with salt-water,but the method of steaming with salt-water is adopted in Guangdong Province.Xia Yanan[33]and other studies found that the dryness toxicity of BuGuZhi can be divided into two aspects:the toxicity to the organs and the damage to the dryness of Yin.The crude BuGuZhi has remarkable dryness.After stri-frying with salt-water,the dryness and toxicity can be alleviated.Wang Zenghui et al.used ultra-high performance liquid chromatography mass spectrometry to measure the chemical constituents of crude BuGuZhi and salt-roasted BuGuZhi[34].The results showed thatpsoralen and isopsoralen were increased after stir-frying with salt-water,while the content of bakuchiol decreased.Tao Yi et al.studied twelve chemical constituents of four kinds of BuGuZhi which are crude,stir-frying with salt-water，stri-frying with wine and stri-frying,and they found that the contents of bakuchiol which damaged the liver and kidney function decreased in stri-frying with wine product and stir-frying with salt-water product of BuGuZhi[35].There are some reports that the key factor to promote the efficiency and attenuation of drugs is the temperature of processing and the time of heating[36].Hu Xin et al.used orthogonal experimental design to optimize the processing technology of BuGuZhi, and finally determined the best process of stir-frying with salt-water method is 20 kg BuGuZhi in 400°C stir-frying for 4-6 minutes until medicinal materials plump up slightly[37].Li Na et al.obtained the best processing technology of BuGuZhi by Doptimal design response surface methodology combined with UHPLC,which is adding 2.1 g salt into 100 g BuGuZhi stir-frying at 80°C for 30 minutes after 12 hours of moistening[38].The traditional processing technology considers that stir-frying with salt-water is the best processing technology,however,Cai Taotao et al.found that stir-frying with salt-water of BuGuZhi could enhance the toxicity [39].The mechanism may be related to the increase of osmotic pressure due to Na+,thus promoting the absorption of the whole components of BuGuZhi in the intestine.Li Hong-fang and other studies found that the effective composition of BuGuZhi in steaming with salt-water was higher than which in stir-frying with salt-water[40].it is necessary to establish a more standardized processing technology to improve the quality of Chinese herbal pieces and the processing technology,which will achieve the purpose of synergism and detoxification.The new processing technology of BuGuZhi can play a synergistic and detoxifying role.Therefore,Xu Nan et al.have put forward the theoreticalhypothesis of "processing transformation",which means ensuring the quality of Chinese herbal slices by monitoring and controlling their chemical reactions and products in the process of processing [41].By processing,the contentand composition of active ingredients in the slices can be transformed to the optimal direction,which can improve the efficacy of the slices or reduce the toxicity.

Study on herbals compatibility in synergistic and attenuated pathways

BuGuZhi has the characteristics ofbitterness,pungency and warmth.The warm-warm compatibility represented by nutmeg can enhance the efficacy and reduce the toxicity.Pan Xin et al.studied the effects of BuGuZhi and nutmeg on warming kidney and spleen,astringent intestines and stopping diarrhea[42].The combination of warm and hot like cinnamon can increase the effect of Fructus Psoralen in warming the interior and dispersing cold.Cold-warm compatibility can reduce toxicity,inhibit its hot and dry medicinal properties,and Rehmanniagluti nosa is cold,which can restrict the warm and dry nature of BuGuZhi.From the perspective of five tastes,the compatibility of acid and pungent can reduce toxicity.Acidic drugs have astringent effect,which can protect Yin blood and restrict the power of dissipation.Fructus Schisandrae in the sishen pill has austerity and astringent effect.BuGuZhi and Fructus Schisandrae can promote the dissolution of active ingredients and inhibit the production of toxic ingredients,which can achieve the purpose of synergism and detoxification.Compatibility of pungent and sweet like Polygonum multiflorum can synergy and attenuated,Sweet can supplement and alleviate the drug property,as the sweet and wet Polygonum multiflorum can restrain the dryness of BuGuZhi.However,Spicy-sweet can also produce Yang Qi,and sometimes it will also increase the toxic effect[43].Ancientliterature "Haiyao Bencao" records:Psoralen Fructus"evilGlycyrrhiza uralensis".The combination of BuGuZhi and Glycyrrhiza uralensis may increase the risk of toxicity.The physique of patients shouldbe identified in clinical application,and the patient whose physique isyin deficiency and internal heat should use cautiously.Zhao Zijing et al.found that there was no significant differencebetween BuGuZhi combined with GanCao(Glycyrrhiza uralensis)and BuGuZhi on liver and kidney function damage[44].At present,there are few researches on the toxic effect of Psoralea corydalis and GanCao,and there is no scientific experimental data to guide clinical practice, so further scientific experimental researches are needed.Deng Pingxiang et al.carried out experimental research with the aqueous extract of BuGuZhi and compound Psoralea Fructus aqueous extract(The compound is purified from psoralen and other Chinese herbs.)[45].The results showed that the aqueous extract of Psoralea Fructus cause hepatocytes injury,and the aqueous extract of compound Psoralea Fructus had no effect on the liver.Cheng Xufeng and others study the dose-effectrelationship ofbone metastasis ofbreast cancer with Psoralen Fructus and SheChuangZi(Cnidii Fructus),which found that 2:2 proportion of SheChuangZi and BuGuZhi could inhibit bone metastasis of breast cancer[46].Zoledrophosphoric acid was used as a control drug,and the result showed that the effect of the two drugs was similar.In addition,the combination of BuGuZhi and cardamom seed can enhance the anti-tum effect and play a synergistic effect[47].Based on the compatibility and application of BuGuZhi,it served as an effective way to reduce toxicity.In the course of mixed decoction,drugs may produce compounds which arebeneficial to enhance the efficacy and reduce the toxicity of drugs.Then it will play the greatest clinical efficacy,at the same time the dryness and toxicity willbe reduced as well.

Research on ways of synergism and attenuation by patter-n identification

Traditional Chinese medicine(TCM)is mostly derived from natural plants,animals and minerals.Under the guiding principles of TCM,pattern identification and flexible prescription application,it has great curative effect in the treatment of many diseases.It generally believes that TCM has less side effects than western medicine.However,with the wide application of TCM in clinical practice,the incidence of adverse reactions is also in an increasing trend.More and more attention has been paid to the occurrence of adverse reactions such as the Xiaochaihu Granule Incident in Japan and the Coptis Incident in Singapore,which are related to unreasonable use of drugs.The key to use drug rationally under the principle of pattern identification in Chinese medicine is the combination of dialectics and rationality.In the Treatise on Febrile Diseases,it is pointed out that"observing the pulse disease,knowing what is wrong and treating it according to syndrome"and doctors should grasp the taboo of medication to achieve"treating cold with heat","treating heat with cold","treating deficiency with tonification"and"treating excess with purgation".Accurate identification is the necessary guarantee for reducing toxicity. Only correlation of all four examinations,identifying the etiology and pathogenesis can we reduce the side effects caused by the improper use of drugs."Toxicity"referring to the bias of drugs[48].The reason why drugs can treat diseases is that they have a certain"bias",which can also become"toxicity",the ancients said,the drugs all have some poisons.The treatment of TCM depends on the accuracy of pattern identification.The unclear diagnosis will lead to improper use of drugs,which can aggravate the disease.Therefore,it is necessary to administer drugs regularly to prevent and treat poisoning reaction caused by accumulation of toxic drugs in the body[49].

Constitution identification and appropriate dosage

Doctors should master the contraindications of drugs and the unsuitable crowd,in which way can we avoid adverse reactions that is owing to unclear constitution identification.And they must prescribe prescriptions according to the common dose provided in the Pharmacopoeia(6-10 g),the first dose should start from a low dose,then increase the dosage gradually and avoid using BuGuZhi alone with long-term and high-dose.In clinical,we should carefully inquire whether patients have medical history of diseases such as liver and kidney.And increase ordecrease dose according to the differences in age,sex and constitution of patients.

Effects of origin and quality of medicinal materials on synergism and attenuation of BuGuZhi

Different origins of Chinese medicinal materials make different drug properties and toxicity.Therefore,when selecting raw materials,we should consider the difference of origins,and different raw materials should be analyzed as well.In recent years,the market of medicinal materials has been chaotic for fake products are emerging in an endless stream.Some illegal traders fake BuGuZhi with Daturae flos seeds,Datura seeds and henbane seeds,which can cause serious toxic reactions after eaten by mistake[50].Dai Yonghaiand others in the theory of toxicity and non-toxicity of traditional Chinese medicine mentioned that the toxicity and non-toxicity is relative,with two sides,and will be transformable under certain conditions[51].Clinicians should try to transform toxicity into non-toxicity and improve the efficacy of drugs.In the process of collecting and storing of Chinese medicinal materials,the quality of it should be checked strictly,and the market circulation of fake and inferior products must be eliminated.

Conclusion and Outlook

As one of kidney-reinforcing drugs are used commonly in clinic,it is mainly used in orthopedics,andrology and other diseases in Chinese herbal decoction.In modern medicine,the extract of BuGuZhi is widely used in clinic,and its clinical value is worth affirming.We have summarized the toxicity of BuGuZhi in recent years and the ways of synergism and detoxification as above.Understanding the toxicity of BuGuZhi can give a better guidance to the clinical safety of medication,which can maximize synergism and detoxification to the most extent.The main toxic effectofBuGuZhiis inducing hepatotoxicity.The experimental results show that the hepatotoxicity occurs only when the BuGuZhi is used in large doses(far more than clinical doses)for a long time.However,the actual clinical cases are not consistent with the experimental results considering that the absorption and metabolism of BuGuZhi components in animal experiments may be different from human body.The difference of individual constitution(sex,age,weight,etc.)may be an inducement of the disease.From the view of traditional Chinese medicine,it is possible that the patients’constitution is mainly internal heat.If the patients’constitution is not clearly recognized,it may encourage dampness and heat.Most of the liver and gallbladder diseases are mainly dampness and heat,which may increase the disorder of liver and gallbladder,then cholestasis may increase.The composition of BuGuZhi is complex and its toxic mechanisms are not clear,and these studies are only confined to animal experiments.So how to enhance efficiency and reduce toxicity becomes the emphasis of future research of BuGuZhi.However,there are few studies on this aspect,a more comprehensive and scientific explanation on toxicity, synergy and detoxification mechanism is needed to guide physicians to use drugs safely.Nonstandard processing is also an importantfactorwhich causes inferiorquality of decoction pieces.As for the circumstances mentioned,the state drug regulatory departments should improve relevant laws and regulations,supervise the quality of raw materials strictly,further standardize the processing technology,and crack down on illegal traders selling counterfeitdrugs.Regularprofessionaltraining of pharmacists should be carried out to minimize the harm to human life and health,which are caused by taking counterfeit or inferior medicines.

Acknowledgements

The authors wish to thank Guowei Zhang(M.D.and Ph.D.,China)for his invaluable assistance.

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