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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 790792 9 pageshttpdxdoiorg1011552013790792

Research ArticleClearance of Free Silica in Rat Lungs by Spraying withChinese Herbal Kombucha

Nai-fang Fu1 Chang-hui Luo1 Jun-cai Wu1 Yan-yan Zheng2 Yong-jin Gan2 Jian-an Ling2

Heng-qiu Liang2 Dan-yu Liang2 Jing Xie2 Xiao-qin Chen2

Xian-jun Li2 Rui-hui Pan2 Zuo-Xing Chen2 and Sheng-jun Jiang1

1 Tropical Crops Genetic Resources Institute Chinese Academy of Tropical Agricultural Sciences Hainan 571737 China2Guangxi Zhuang Autonomous Region Institute for Chemical Toxicity Testing Guangxi Zhuang Autonomous Region Academy forthe Prevention and Treatment of Occupational Disease Guangxi 530021 China

Correspondence should be addressed to Xiao-qin Chen 350061439qqcom and Sheng-jun Jiang 18708938111139com

Received 25 March 2013 Accepted 4 June 2013

Academic Editor Tzeng-Ji Chen

Copyright copy 2013 Nai-fang Fu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The effects of spraying with kombucha and Chinese herbal kombucha were compared with treatments with tetrandrine in a ratsilicosis model Silica dust (50mg) was injected into the lungs of rats which were then treated with one of the experimentaltreatments for a month The rats were then killed and the effects of the treatments were evaluated by examining the extent andseverity of the histopathological lesions in the animalsrsquo lungs measuring their organ coefficients and lung collagen contentsdetermining the dry and wet weights of their lungs and measuring the free silica content of the dried lungs In addition lavagewas performed on whole lungs taken from selected rats and the numbers and types of cells in the lavage fluid were countedThe most effective treatment in terms of the ability to reduce lung collagen content and minimize the formation of pulmonaryhistopathological lesions was tetrandrine treatment followed by Chinese herbal kombucha and non-Chinese herbal kombuchaHowever the lavage fluid cell counts indicated that tetrandrine treatment had severe adverse effects on macrophage viability Thiseffect was much less pronounced for the kombucha and Chinese herbal kombucha treatments Moreover the free silica levels inthe lungs of animals treated with Chinese herbal kombucha were significantly lower than those for any other silica-exposed groupThese preliminary results indicate that spraying with Chinese herbal kombucha preparations can effectively promote the dischargeof silica dust from lung tissues Chinese herbal kombucha inhalation may thus be a useful new treatment for silicosis and otherpneumoconiosis diseases

1 Introduction

Silicosis is a pneumoconiosis disease caused by exposure tovarious forms of silica dust including crystalline silica andamorphous silica dust [1] It is caused by the inhalation offine silica particles which are deposited in the lungs andingested by macrophages This triggers an immune responsethat stimulates the production of collagen around the particleresulting in the formation of nodular lesions that obstructthe airways Occupational exposure to silica dust and theresulting health problems are major public health issues inboth developed and developing countries [2] It was recentlyreported that 23 million workers in China have been exposedto crystalline silica dust and that more than ten million

workers in India are at risk of exposure [3 4] Similarly morethan 17 million workers in the USA and more than 3 millionworkers in Europe are likely to have been exposed to silicadust in the workplace [5 6] Silicosis has a range of adverseeffects on health including an increased susceptibility totuberculosis lung cancer and pulmonary heart diseaseTheseproblems are exacerbated by the lack of an effective treatmentfor the condition

Kombucha is a drink made by fermenting sugar and teaextracts with kombucha It is rich in acetic acid bacteriayeast probiotics acetic acid and other organic acids that arebeneficial to human health and can inhibit the growth ofharmful bacteria It has proven to be a good treatment foratrophic gastritis and gastric ulcer disease and can also help

2 Evidence-Based Complementary and Alternative Medicine

regulate blood pressure slow aging and prevent and treatvarious diseases [7] Kombucha contains two notable groupsof microorganisms Gluconoacetobacter xylinus (Xylinum)and yeasts Xylinum secretes bacterial cellulose through holesin its cell walls Interestingly kombucha cultures producebacterial cellulose more efficiently than cultures of Xylinumalone [8 9] The bacterial cellulose produced by kombuchacultures has a number of properties that make it potentiallyuseful inmedical applications including good biocompatibil-ity thinness (the cellulose sheets are typically only 01micronsthick) and a high specific surface areaThese propertiesmeanthat it functions as a nanoscale functional material with adefined three-dimensional structure and a large number ofsurface-exposed hydroxyl groups which allow it to formstrong noncovalent bonds with water and also a wide range ofions and organic compounds [10] It has been demonstratedthat bacterial cellulose efficiently adsorbs numerous toxicheavy metal ions including Cu2+ Pb2+ Hg2+ and Cd2+ Inaddition it adsorbs a range of nonmetallic toxins such ashistamines ammonia and NO2minus and formaldehyde [11ndash16]We therefore hypothesized that if kombucha were sprayedinto lung tissues the bacterial cellulose produced by theculturemight adsorb dust and protein precipitates that wouldotherwise cause the symptoms of silicosis

Chinese herbal kombucha preparations can be madeby fermenting extracts of various plants with a kombuchaculture If the plants used in these preparations containbiologically active substances the resulting Chinese herbalkombucha may combine the beneficial effects of the kom-bucha itself with those of the plants A range of plants andherbs can be used for this purpose including licorice Siratiagrosvenori mangosteen and chrysanthemum Licorice isknown to have numerous pharmacological effects and iswidely used in traditional medicine It contains a range ofbiologically active compounds including glycyrrhizin gly-cyrrhetinic acid and the licorice flavonoids Both licorice andthe isolated active compounds are known to have antitussiveand expectorant properties Moreover it is effective againstasthma and offers protection against respiratory pathogens[17 18] Siratia grosvenori is a specificChinese plant belongingto the family Cucurbitaceae When dried its fruit is verysweet and has a cooling effect It is an important ingredientof ldquocooling drinksrdquo and is considered to be beneficial forrelieving conditions characterized by high body tempera-tures such as inflammation It is used in traditional Chinesemedicine to treat chest pains dry coughs sore throats andaphonia [19] Mangosteen aqueous extracts have been shownto significantly reduce the frequency of coughing in guineapigs treatedwith citric acid or capsaicin and inhibits coughingcaused by mechanical stimulation [20] Chrysanthemumis an important medicinal plant that has various pharma-cological effects Among other things it functions as anantibacterial anti-inflammatory vasodilatory and antitumoragent as well as reducing blood pressure and acting as anantioxidant [21] Teas made from the buds of the camelliaplant containmany bioactive compounds including polyphe-nols tea polysaccharides alkaloids vitamins and aminoacids as well as various essential metal ions As a result theyhave a wide range of pharmacological activities including

anti-inflammatory antioxidant and antithrombosis effectsas well as being useful in the treatment of diabetes and forreducing blood pressure [22 23]

In light of these facts we sought to investigate the utilityof kombucha and various Chinese herbal kombuchamixturesfor the treatment of silicosis and other diseases arising fromthe presence of dust in the lungs To this end rats wereexposed to silica dust via tracheal injection and then sprayedwithChinese herbal andnon-Chinese herbal kombucha solu-tionsThe results of the kombucha treatments were comparedto those observed following treatment with tetrandrine acompound that is known to be useful for mitigating thesymptoms of silicosis [24] The different treatments wereevaluated in terms of their effects on lung anatomy collagenlevels in the lung tissues of the experimental animals andtoxicity

2 Materials and Methods

21 Materials Silica dust (99 particle diameter 05ndash10 120583mwith 80 of particles having diameters of 1ndash5 120583m) waspurchased from Sigma Aldrich Tetrandrine was purchasedfrom the Zhejiang Zhongyi Pharmaceutical Co Ltd A kit formeasuring collagen (hydroxyproline) levels was purchasedfrom the Nanjing Jiancheng Bioengineering Institute

22 Preparation of the Kombucha Mixtures Kombuchastrains were purchased from the Beijing Institute of FoodResearch

A kombucha stock solution was prepared according tothe method of Ai-jun Lv [23]The Chinese herbal extract wasprepared bymixing tea (02ww) licorice (05ww) driedSiratia grosvenori fruit (05 ww) and wild chrysanthemum(02 ww) in water and boiling the resulting mixtures for20minThe boiled solution was then filtered cooled to below30∘C and mixed with a 20 dilution of the kombucha stocksolution After fermenting at 30∘C for 2 weeks the Chineseherbal kombucha solution was considered ready for use inexperiments

23 Experimental Animals Test animals were provided bythe Hunan Slack Jingda experimental animal company(License number SCXK Hunan) The animals used wereSpecific Pathogen Free (SPF) Sprague-Dawley (SD) rats thathad been held under quarantine for 5 days

24 Tracheal Injection of Method for the Contamination ofSilica Dust [25ndash27] A 50mgmL standard suspension ofquartz (silica) dust in saline was prepared Prior to trachealinjection samples of the stock solution were autoclaved andthen mixed with mycillin (4000UmL) The experimentalanimals were then anaesthetized under sterile conditions andsubjected to intratracheal injection with 1mL of the sterilemycillin-containing silica dust suspension in lungs

25 Experimental Groups and Treatments The 150 experi-mental animals weighed 180ndash220 g each and were randomly

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Experimental groups and treatments

Group Treatment

Chinese herbal kombucha treatment groupStarting four days after being injected with silica the rats were sprayed withChinese herbal kombucha Each rat was sprayed 20 times in the morning and 20times in the afternoon for five consecutive days per week over a four-week period

Kombucha treatment groupStarting four days after being injected with silica the rats were sprayed withnon-Chinese herbal kombucha Each rat was sprayed 20 times in the morning andin the afternoon for five consecutive days per week over a four-week period

Tetrandrine treatment group The rats were treated with tetrandrine by lavage Each rat was dosed with 15mgthree times per week for four weeks

Positive control groupStarting four days after being injected with silica the rats were sprayed with a 1 gLsolution of NaCl Each rat was sprayed 20 times in the morning and 20 times in theafternoon for five consecutive days per week over a four-week period

Negative control groupSynchronously operated with the positive control group at the fourth day ofinstilled with silica Each rat was sprayed 20 times continuously once in themorning and once in the afternoon for five consecutive days for four weeks

allocated to five different groups of 30 animals each 15 malesand 15 females (see Table 1)

26 Measurement of Organ Coefficients and the Wet andDry Weights of Lung Tissues At the end of the treatmentperiod the animals were killed by arterial bloodlettingTheir hearts livers spleens kidneys and other organs andtissues were then immediately removed and weighed andthe organ coefficient for each organ was calculated (organcoefficient = organ weightbody weight times 100) The tracheaand lungs were then removed and separated and the lungswere stripped of their connective tissue The connectivetissues were soaked in water and the wet lung weight (119872)was measured A 05 g lung tissue sample was then immersedin acetone for three days for degreasing and then cut intopieces baked in an oven at 105∘C for 12 h and weighed todetermine its dry weight (119898) The dry weight of the lung wasthen calculated as 2119898 times119872

27 Determination of Total Lung Collagen (Hydroxyproline)[27] Lung homogenates were prepared and their hydrox-yproline content was determined using the chloramine-Tmethod according to the instructions provided with the kit

28 Counting and Classification of Cells in Lung Lavage FluidThe rats were killed by arterial bloodletting from the groinThe trachea was then removed and a V-shaped opening wasmade at the 13 point of the lower trachea One end of a smallplastic hose was fitted with an eight-gauge needle and theother end was inserted into the V-shaped opening and ligatedto the lung using fixed lines 5mL of saline was taken up ina syringe which was then affixed to the needle on the endof the tubing The saline was slowly injected into the alveoliand then slowly withdrawn to yield approximately 3mL ofrecovered liquid The cells within this recovered liquid werecounted and classified under a microscope

29 Pathological Analysis of Lung Tissues First the gross lungmorphology was observed The lung tissue was then fixed

using formalin Conventional paraffin sections were takenand stained with hematoxylin-eosin The stained sectionswere then examined using an optical microscope (OlympusBX43) Pathological changes and nodules in the lung tissueswere graded and stained lung tissue collagen fiber andreticular fiber hyperplasia were observed with Model BX43OlympusOptical microscopyThe results obtained were eval-uated according to the diagnostic criteria for pneumoconiosisspecified in the national occupational health standards of thePeoplersquos Republic of China (GBZ25-2002)

210 Determination of the Free Silica Contents of WholeLung Samples Using the Pyrophosphate Method [28] Silicalevels in lung samples were measured using the method fordetermining silica levels in dust samples described inChinesenational standardGBZT 1924-2007 (ldquoDetermination of dustin the air of workplace-Part 4 Content of free silica industrdquo) Fresh rat lung sampleswere degreased dried and thencrushed Samples of the crushed material (010 g) were thenanalyzed using the previous method

3 Statistical Analysis

All data were recorded in the form 119909 plusmn 119904 where 119909 is themean value from a given number of observations and 119904 isthe associated standard deviation The 119905-test was performedusing the SPSS software package and used to analyze thesignificance of differences between groups using a thresholdvalue of 119875 lt 005

4 Results

41 Clinical Manifestations of Silica Exposure In the periodimmediately following their injection with silica dust the ratsexhibited symptoms of dyspnea did not eat much and weresluggish In the early stages of the treatment period noneof the silica-exposed groups exhibited any significant clinicalabnormalities In the later stages of the treatment periodindividual rat weights decreased significantly Four deathsoccurred in total one in the kombucha treatment group one


Table 2 Changes in the weight of dust-exposed rats over the course of the experimental period (unit g)

Treatment 10 days before silicaexposure

2 days aftertreatment




Chinese herbal kombucha 1640 plusmn 71 2028 plusmn 139998779998779

2251 plusmn 189 2401 plusmn 248 2531 plusmn 293

Kombucha 1636 plusmn 68 1971 plusmn 112998779998779

2224 plusmn 179998779

2381 plusmn 220 2498 plusmn 290

Tetrandrine 16487 plusmn 62 2036 plusmn 128998779998779


Positive control 1656 plusmn 59 2005 plusmn 126998779998779

2208 plusmn 190998779

2372 plusmn 236 2484 plusmn 267

Negative control 1647 plusmn 66 2177 plusmn 178 2342 plusmn 238 2487 plusmn 290 2632 plusmn 311

998779Value is significantly different to that for the negative control group (119875 lt 005)998779998779Value is significantly different to that for the negative control group (119875 lt 001)

Table 3 The influence of the various treatments on the organ coefficients of rats exposed to silica dust

Treatment Heart Liver Spleen Kidney LungChinese herbal kombucha 029 plusmn 002

lowast335 plusmn 058 025 plusmn 003 068 plusmn 005 182 plusmn 080998779

Kombucha 028 plusmn 003lowast

329 plusmn 059 025 plusmn 005 063 plusmn 005 206 plusmn 028998779

Tetrandrine 033 plusmn 004998779


Positive control 030 plusmn 002lowast998779


Negative control 028 plusmn 002lowast


lowastValue differs significantly from that for the tetrandrine treatment group (119875 lt 005)998779Value differs significantly from that for the negative control group (119875 lt 005)

in the tetrandrine treatment group one in the positive controlgroup and one in the negative control group No deathsoccurred in the Chinese herbal kombucha treatment group

42 Changes in RatWeight following Silica Exposure The ratswere weighed once per week during the treatment period Ascan be seen in Table 2 there were no significant differencesbetween the average weights for each group prior to theirinjection with silica One week after exposure to the dust theweight of the negative control group was significantly higherthan that of the groups exposed to silica (119875 lt 001) By thesecond week of the treatment period the weights of the ratsin the Chinese herbal kombucha and tetrandrine treatmentgroups were not significantly different to those of the negativecontrol group The weights of the rats in the kombuchatreatment group and the positive control group also increasedbut remained significantly lower than those of the negativecontrol animals (119875 lt 005) This indicates that the Chineseherbal kombucha and tetrandrine treatments both promotedthe regaining of weight following silica exposure

43 Organ Coefficients after Silica Exposure At the end ofthe treatment period the hearts livers spleens lungs andkidneys of the rats in each group were removed and weighedand the corresponding organ coefficients were calculatedThe results (Table 3) indicate that there were no significantdifferences between the negative control group and any ofthe silica-exposed groups in terms of the organ coefficientsfor the liver spleen or kidney However the lung coefficientsfor all of the dust-exposed groups were significantly greater(119875 lt 005) than those for the negative control group Therewere no significant differences between the lung coefficientsfor the various silica-exposed groups

The lung coefficient data shown in Table 3 demonstratethat the Chinese herbal kombucha and tetrandrine treat-ments inhibited the hyperblastosis of the lung tissue causedby silica exposure Table 3 also shows that the heart coefficientfor the negative control group was significantly lower thanthose for the positive control group and the tetrandrinetreatment group (119875 lt 005) This suggests that both silicaexposure and oral tetrandrine treatment have adverse effectson cardiac health

However the heart coefficients for the kombucha andChinese herbal kombucha treatment groups were signifi-cantly lower (119875 lt 005) than those for the tetrandrinetreatment group and were not significantly different fromthose for the negative control group This indicates thatkombucha and Chinese herbal kombucha are effective atmitigating the cardiotoxic effects of inhaling silica dust

44 Cell Counts in Lung Lavage Fluid from Silica-ExposedRats The negative control group had the lowest lung lavagefluid cell count (Table 4) averaging 0308 times 109 cellsmLTetrandrine treatment yielded the highest average lavage fluidcell count (720 plusmn 1362 times 109 cellsmL) one sample fromthis group had a count of 35 times 109 cellsmL The cell countsin the lavage fluid from rats in other treatment groups were246 plusmn 178 times 10

9 cellsmL for the Chinese herbal kombuchagroup 119 plusmn 104 times 109 cellsmL for the kombucha groupand 112 plusmn 075 times 109 cellsmL for the positive control groupBecause of the considerable variation within each treatmentgroup there were no significant between-group differences

In general the greater the total number of cells within thelavage fluid the more severe the case of silicosis In conjunc-tion with the finding that tetrandrine treatment suppresseshyperplasia in the lungs (Table 3) the high numbers of cells


Table 4 The effects of the various treatments on the cell counts in lung lavage fluid from rats exposed to silica

Treatment Total cells (times109mL) Cell type ()N L M

Positive control 112 plusmn 075 0685 plusmn 0004lowast

0247 plusmn 0067lowast


Negative control 031 plusmn 019 0272 plusmn 0226 0040 plusmn 0051 0692 plusmn 0266

Chinese herbal kombucha 246 plusmn 178 0633 plusmn 0320lowast



Kombucha 119 plusmn 104 0718 plusmn 0138lowast



Tetrandrine 720 plusmn 1362 0600 plusmn 0192lowast



lowastValue differs significantly from that for the negative control group (119875 lt 005)

in the lung lavage fluid of the tetrandrine treatment groupindicate that tetrandrine is a poor therapeutic agent due toits toxicity towards the tissues of the lung The counted cellsin the lung lavage fluid for each treatment group were sortedby type (Table 4) It was found that lymphocytes (L) neutralcells (N) and giant divinatory cells (M) accounted for the vastmajority of these cells

At the end of the treatment period the most abundantcell type in the lavage fluid of the negative control groupwas M cells followed by N and then L cells Converselythe lavage fluid of the dust-exposed groups was dominatedby N cells followed by L and then M cells There weresignificant differences in the proportions of the different celltypes between the negative control group and the silica-exposed groups (119875 lt 005) M cells are phagocytes that areimportant for lung healthTheir numbers are greatly reducedby dust exposure causing the relative abundance of N cells toincrease

45 Hydroxyproline Levels in the Lungs of Silica-Exposed RatsHydroxyproline assays were performed on lung samplestaken from rats killed one week after the end of the treatmentperiod (30 days after the onset of treatment) and fromanimalskilled three weeks after the end of the treatment period (50days after the onset of treatment) The results for the day 30group are shown in Table 5 The only treatment group withhydroxyproline levels thatwere significantly different to thosefor the negative control group was treated with non-Chineseherbal kombucha (119875 lt 005) For the day 50 group thehydroxyproline levels declined in the following order posi-tive control gt kombucha treatment group gt Chinese herbalkombucha treatment gt tetrandrine treatment gt negativecontrol group All of the silica-exposed groups other than thetetrandrine treatment group had hydroxyproline levels thatwere significantly greater than those for the negative controlgroup (119875 lt 005) Since lung hydroxyproline levels are abiochemical indicator of pulmonary fibrosis this suggeststhat tetrandrine is effective at inhibiting fibrosis

In all cases the hydroxyproline levels measured for theday 50 animals were substantially greater than those forthe day 30 animals (Table 5) Moreover the between-groupdifferences for the day 50 animals were more significant thanthose for the day 30 animals Both of these results indicatethat lung fibrosis becomes more severe over time

Table 5 The impact of the various treatments on hydroxyprolinelevels in the lungs of silica-exposed rats

Treatment Number of animals Hydroxyproline level (mgglowast)Day 30 Day 50

ChineseherbalKombucha

6 045 plusmn 018 073 plusmn 022998779

Kombucha 6 050 plusmn 014998779075 plusmn 012

998779

Tetrandrine 6 037 plusmn 009 056 plusmn 017

Positivecontrol 6 045 plusmn 009 079 plusmn 028

998779

Negativecontrol 6 034 plusmn 005 040 plusmn 006

998779Value differs significantly from that for the negative control group (119875 lt005)lowastMilligrams of hydroxyproline per gram of lung tissue

46 Lung Pathology Visual inspection of the pathologicalsections of the lungs of the experimental rats (Figures 1(a)ndash1(e)) indicated that there were no abnormalities among thenegative control group However significant partial lungconsolidation was observed for the kombucha treatmentgroup the Chinese herbal kombucha treatment group andthe positive control group The lesions in the sections takenfrom animals treated with non-Chinese herbal kombuchawere particularly heavy whereas the sections from animalstreated with Chinese herbal kombucha were more similarto those for the positive control group The sections fromthe tetrandrine treatment group exhibited less extensive lungconsolidation and more evidence of lung inflammation

47 Wet and Dry Lung Weights Among the experimentalgroups the wet lung weight decreased in the followingorder kombucha treatment group gt positive control group gtChinese herbal kombucha treatment group gt tetrandrinegroup gt negative control group (Table 6) The differencebetween the values for the tetrandrine treatment group andthe Chinese herbal kombucha treatment group was notstatistically significant but that between the values for thepositive control group and the tetrandrine group (119875 lt 005)The dry lung weights for the various groups decreased in thesame order as that for the wet lung weights Based on these


Table 6 The effects of the tested treatments on wet and dry lung weights in silica-exposed rats

Treatment Total wet lung weight (g) Total dry lung weight (g) Total lung moisture content ()Negative control 142 plusmn 043 034 plusmn 009 7615 plusmn 131

Chinese herbal kombucha 379 plusmn 093 090 plusmn 021 7614 plusmn 110

Kombucha 478 plusmn 114 112 plusmn 018 7619 plusmn 152

Tetrandrine 267 plusmn 026 075 plusmn 006 7196 plusmn 074

Positive control 438 plusmn 085 104 plusmn 014 7614 plusmn 163

Table 7 The effects of the tested treatments on dust removal from the lungs of silica-exposed rats

TreatmentWhole lung freesilica content

(mg)

Free silica removedfrom the lung (mg)

Free silica clearance rate forthe whole lung ()

Silica discharge rate for thewhole lung (mgd)

Negative control 20 plusmn 10 mdash mdash mdashChinese herbal kombucha 380 plusmn 210

lowast1397 plusmn 2065 2792 plusmn 4130 047 plusmn 069

Kombucha 540 plusmn 50 minus246 plusmn 468 minus492 plusmn 936 minus008 plusmn 016

Tetrandrine 520 plusmn 120 minus018 plusmn 1178 minus035 plusmn 2355 minus001 plusmn 039

Positive control 440 plusmn 60 581 plusmn 591 1163 plusmn 1183 025 plusmn 020

mdash because the rats in the negative control group were not exposed to silica the analysis was not performed in this caselowastValue differs significantly from that for the tetrandrine treatment group (119875 lt 005)

results it was found that the water content of the lungs of ratstreated with tetrandrine was relatively low (7196 plusmn 074)but that all other groups had similar lung water contentsThisimplies that tetrandrine treatment causes some level of tissuedehydration

Intrapulmonary levels of free silica weremeasured in lungsamples from animals in each of the experimental groupsThe highest free silica levels occurred in the tetrandrinetreatment group (520 plusmn 120mg) The value for the Chineseherbal kombucha group (380 plusmn 210) was significantly (119875 lt005) lower than that for both the tetrandrine group andthe positive control group (440 plusmn 60mg) Interestinglythe free silica level for the non-Chinese herbal kombuchatreatment group was relatively high (540 plusmn 50mg) Theseresults suggest that treatment with Chinese herbal kombuchastrongly promotes the discharge of free silica dust whereastreatment with tetrandrine or non-Chinese herbal kombuchadoes not promote dust emission and may in fact causesome level of dust enrichment Over the 30-day treatmentperiod the rate of silica discharge from the lungs of the ratstreated with Chinese herbal kombucha was 047 plusmn 069mgd(Table 7)

5 Discussion

Silica dust is the main pathogenic factor of silicosis Conse-quently the development of effective methods for removingsilica dust from the lungs will be essential for effectivelytreating this disease This study explored the scope forusing Chinese herbal and non-Chinese herbal kombuchapreparations as dust-removing probiotic agents for treatingsilicosis and related conditions At present silicosis is treatedusing drugs such as oxypovidine tetrandrine and aluminumcitrate which only alleviate the symptoms of the disease

there is currently no cure Tetrandrine is the most widelyused drug for treating pneumoconiosis in China and there isstrong evidence that it directly or indirectly inhibits collagengene transcription thereby reducing collagen synthesis inthe affected tissues Long-term use of tetrandrine can reducethe severity of the respiratory symptoms of silicosis and thenumber of lung infections suffered as well as improvinglung function However it can also cause skin discolorationand itching Approximately 20 of all patients treatedwith tetrandrine experience sodium deficiency bloating andapproximately 98 experience impaired liver function [2930] The results presented herein suggest that in addition tothese effects tetrandrine may be toxic to cardiac tissue andcause lung dehydration the latter effect may be related to itsknown tendency to cause skin dehydration

Our results indicate that tetrandrine treatment can sup-press the formation of collagen in lung tissues However thecell counts in lung lavage fluid from tetrandrine-treated ratssuggest that it has cytotoxic effects in the lungs and mayinhibit the discharge of silica dust This is consistent with thepoor outcomes and severe side effects observed for patientsthat have been treated with tetrandrine for extended periodsof time [29 30] As such it may not be appropriate to usethe inhibition of collagen synthesis in lung tissue as the mainindicator of effectiveness when evaluating the performance ofdrugs for the treatment of silicosis

Aside from medication the most common treatmentfor pneumoconiosis-type diseases such as silicosis is largevolume whole lung lavage This method involves repeatedlyflushing the lungs with saline under intravenous anesthe-sia together with mechanical ventilation to remove thepathogenic factor [31] However there are several groupsof patients that are not suitable for large volume wholelung lavage including those with (1) conditions that affect


(a) (b)

(c) (d)

(e)

Figure 1 Pathological analysis of lung tissues from rats exposed to silica (400x) (a) Chinese herbal kombucha treatment group (b) kombuchatreatment group (c) tetrandrine treatment group (d) positive control group (e) negative control group

blood clotting (2) severe tracheal or bronchial deformities(3) illnesses or dysfunctions of the heart brain liver kidneysor other major organs (4) cancers or compromised immunesystems (5) active tuberculosis (6) pulmonary bullae espe-cially subpleural bullae greater than 2 cm in diameter (7)severely low pulmonary function and (8) severe emphysemaor related conditions [31] An analysis of 5000 cases inwhich large volume whole lung lavage was performed totreat pneumoconiosis or some other lung disorders indicatedthat the short-term effects (1ndash3 years) of the treatment aregood but its long-term effects (6ndash7 years) are not significantIn most cases lavage causes reductions in chest tightness(reported by 99 of patients) chest pain (reported by 86of all patients) and shortness of breath (reported by 88 ofall patients) with these beneficial effects lasting for around

three years The average amount of dust cleared from eachlung was 3000 to 5000mg including 70ndash200mg of freesilica However extensive removal of pulmonary alveolarmacrophages was also observed [32] While some of the dustand other foreign material is removed from the lungs bylavage the process can cause significant secondary damageresulting in complications such as tuberculosis Additionallylung lavage is expensive and much of the cost is borneby the patient since most pneumoconiosis patients haveeconomic difficulties it would be desirable to find a less costlyalternative

Our experiments using silica-exposed rats demonstratedthat spraying with Chinese herbal kombucha preparationshas no toxic side effects and effectively promotes the dis-charge of silica dust from the lungs The silica dust exhaust


rate for rats (average body weight 0200 kg) treated withChinese herbal kombucha was 04mgday Simple linearextrapolation suggests that if a human with a body weightof 65 kg were subjected to the same treatment the corre-sponding rate of silica removal would be 130mgd Ratspassively accept the aerosol therapy during the test buta human undergoing treatment would actively inhale theChinese herbal probiotic It is therefore possible that theresults achieved in clinical trials might be even better thanthose observed with the rat model

The average amount of dust cleared fromeach lung duringlavage is 3000sim5000mg Based on the results obtained in thiswork it would require 23ndash38 days of spraying with Chineseherbal kombucha to achieve a similar effectThis is consistentwith the results obtained when a single pneumoconiosispatient was treated by spraying with Chinese herbal kom-bucha for threemonths (see the case report presented in Sup-plementary Appendix 1 in Supplementary Material availableonline at httpdxdoiorg1011552013790792) The patientexperienced significant reductions in the severity of hissymptoms within a month and X-rays taken at the end of thetreatment period demonstrated that the treatment had sig-nificant beneficial effects on his pulmonary health BecauseChinese herbal kombucha preparations have no toxic sideeffects and can be also used to treat TB patients and thosewith cardiopulmonary dysfunctions they could potentiallyreplace lung lavage as a treatment for pneumoconiosis

It has been demonstrated that the amount of dust in thelungs of pneumoconiosis sufferers ranges from 0 to 60 gramsBased on the systemused to classify cases of pneumoconiosisfirst stage cases occur when the lungs contain 0ndash15 g ofdust this causes dust reticulocyte fibrosis Second stagecases (15ndash30 g dust) are characterized by the appearanceof mixed nodules due to reticulocyte fibrosis Third stagecases (30ndash60 g of dust) are characterized by the appearanceof converged fibrosis nodules [33] Given a dust dischargerate of 130mgd 115 days of treatment with Chinese herbalkombuchawould be required to discharge the bulk of the dustin first stage cases 231 days would be required to treat secondstage cases and 461 days of treatment would be requiredfor third stage cases A treatment cycle of around one yearshould thus be sufficient to treat patients with stage I orII pneumoconiosis However the lung X-rays in the single-patient case study (see Supplementary Appendix 1) showedthat significant quantities of dust were still present within thelungs after three months of treatment so 2-3 treatment cyclesmay be required for the complete removal of dust from thelungs in some cases Even if the treatment only removed dustfrom the surface tissues of the pulmonary alveolae significantimprovements in lung function would be achieved Howeverlonger treatment periods may be required to clear deep-seated dust from the lungs

In this work two probiotic mixtures were used to treatsilicosis Interestingly only the Chinese herbal kombuchapreparations had unambiguously beneficial effects on dustemission Non-Chinese herbal kombucha did not promotedust emission so it appears that the combination of Chineseherbal extracts and the kombucha culture has advanta-geous synergistic effects The plant species used to prepare

the Chinese herbal kombucha are known to have antitussiveexpectorant and antiasthma function as well as protectingagainst respiratory pathogens Based on these results theyalso seem to promote the health of pulmonary tissues anddust emission mediated by the cilia Kombucha culturescontain two groups of symbiotic microbes xylinum andyeasts Xylinum generates bacterial cellulose from the ethanolproduced by the yeast bacterial cellulose is an efficientadsorbing agent that will adhere to dust particles and othersubstances thereby facilitating their removal via expectora-tion

It should be noted that the treatments examined in thiswork were only applied over a period of one month Thisis relatively short and it would be desirable to study theeffects of treatment with Chinese herbal kombucha over alonger period of time In addition the composition of theChinese herbal kombucha mixture has not been optimizedto maximize its therapeutic effect and it is likely that moreextensive research in this area would result in the identifica-tion of more potent mixtures Finally it would be desirableto determine the precise mechanisms by which treatmentwith Chinese herbal kombucha promotes the removal of dustfrom the lungs Overall however the results presented inthis work represent the first effective use of a Chinese herbalprobiotic to promote the emission of dust from the lungs andthe alleviation of inflammation in cases of silicosisThis couldhave significant consequences for the treatment of silicosisand other pneumoconiosis diseases and more generally fortreating conditions involving inflammation of the lungs suchas lung protein deposition psychosis It is very easy toproduce and use the Chinese herbal Kombucha Thereforethe Chinese herbal kombucha would help to globally removesilicosis pneumoconiosis and similar diseases in the futures

Acknowledgments

This researchwas supported in part by earmarked funds fromthe China Agriculture Research System (CARS-32-hnjsj)and the basic scientific fund of the Tropical Crops GeneticResources Institute at the Chinese Academy of TropicalAgriculture Science (009)

References

[1] L M Costantini R M Gilberti and D A Knecht ldquoThephagocytosis and toxicity of amorphous silicardquo PLoS ONE vol6 no 2 Article ID e14647 2011

[2] W Chen Y Liu H Wang et al ldquoLong-term exposure to silicadust and risk of total and cause-specific mortality in Chineseworkers a cohort studyrdquo PLoSMedicine vol 9 no 4 Article IDe1001206 2012

[3] Ministry of Health of the Peoplersquos Republic of China AnnualHealth Statistical Report in 2009 Ministry of Health of thePeoplersquos Republic of China Beijing China 2009

[4] World Health Organization Global Occupational Health Net-work Elimination of Silicosis Gohnet Newsletter 12 WorldHealth Organization Global Occupational Health NetworkGeneva Switzerland 2007


[5] US National Institute for Occupational Safety and HealthHealth Effects of Occupational Exposure to Respirable CrystallineSilica US Department of Health and Human Services Wash-ington DC USA 2002

[6] T Kauppinen J Toikkanen A Savela et al ldquoOccupationalexposure to carcinogens in the European Unionrdquo Occupationaland Environmental Medicine vol 57 no 1 pp 10ndash18 2000

[7] C J Greenwalt K H Steinkraus and R A Ledford ldquoKom-bucha the fermented tea microbiology composition andclaimed health effectsrdquo Journal of Food Protection vol 63 no7 pp 976ndash981 2000

[8] P Ross R Mayer and M Benziman ldquoCellulose biosynthesisand function in bacteriardquo Microbiological Reviews vol 55 no1 pp 35ndash38 1991

[9] S J Tang X X Yang and F Hong ldquoProduction of bacterialcellulose by Kombuchardquo Journal of Cellulose Science and Tech-nology vol 20 pp 40ndash45 2012

[10] C L Zhu F Li and Q S You ldquoPreparation of nanometerbiomaterial bacterial cellulose and observation of its ultra-structurerdquo Journal of Biomedical Engineering Research vol 27pp 287ndash290 2008

[11] M E Argun S Dursun C Ozdemir and M Karatas ldquoHeavymetal adsorption by modified oak sawdust thermodynamicsand kineticsrdquo Journal of Hazardous Materials vol 141 no 1 pp77ndash85 2007

[12] S Chen Y Zou Z Yan et al ldquoCarboxymethylated-bacterialcellulose for copper and lead ion removalrdquo Journal of HazardousMaterials vol 161 no 2-3 pp 1355ndash1359 2009

[13] T Oshima K Kondo K Ohto K Inoue and Y Baba ldquoPrepa-ration of phosphorylated bacterial cellulose as an adsorbent formetal ionsrdquo Reactive and Functional Polymers vol 68 no 1 pp376ndash383 2008

[14] M Lu and X H Guan ldquoAdsorption mechanism of bacterialcellulose to heavymetal ionsrdquoChemical Engineering vol 40 pp29ndash33 2012

[15] H Zhou D P Sun C L Zhu and C Y Xu ldquoStudy on theadsorption of noxious substances in human body by bacterialcelluloserdquo Journal of Cellulose Science and Technology vol 17pp 41ndash46 2009

[16] Y Q Wang and Q S Zhang ldquoStudy on the absorption propertyfor formaldehyde by bacterial celluloserdquo Jiangsu Constructionvol 3 pp 67ndash70 2011

[17] M F Zhang and Y Q Shen ldquoEffects of licorice and its extractson respiratory systemrdquo Drugs amp Clinic vol 25 pp 262ndash2672010

[18] M Menegazzi R Di Paola E Mazzon et al ldquoGlycyrrhizinattenuates the development of carrageenan-induced lung injuryin micerdquo Pharmacological Research vol 58 no 1 pp 22ndash312008

[19] Y Chen and E L Jia ldquoThe research progress of the chem-ical constituents and pharmacological action of Momordicagrosvenorirdquo Pharmaceutical Journal of Chinese Peoplersquos Libera-tion Arm vol 27 pp 171ndash174 2011

[20] J Li P B Li and G J Yuan ldquoThe antitussive effect of waterextrat of grosvenormomordica fruitrdquo Journal ofHainanMedicalCollege vol 14 pp 16ndash18 2008

[21] A Rajic T Akihisa M Ukiya et al ldquoInhibition of trypsinand chymotrypsin by anti-inflammatory triterpenoids fromCompositae flowersrdquo Planta Medica vol 67 no 7 pp 599ndash6042001

[22] C-L Shen J K Yeh J J Cao O L Tatum R Y Dagda andJ-S Wang ldquoSynergistic effects of green tea polyphenols andalphacalcidol on chronic inflammation-induced bone loss infemale ratsrdquo Osteoporosis International vol 21 no 11 pp 1841ndash1852 2010

[23] A Lu J Yin X Hu P Zhang and B Hu ldquoA study onmorphology of tea fungus and inhibition effect of its culturefluidrdquo Journal of Xuzhou Normal University vol 22 pp 73ndash752004

[24] Q-M Xie H-F Tang J-Q Chen and R-L Bian ldquoPharma-cological actions of tetrandrine in inflammatory pulmonarydiseasesrdquo Acta Pharmacologica Sinica vol 23 no 12 pp 1107ndash1113 2002

[25] W Faxuan Z Qin Z Dinglun et al ldquoAltered microRNAsexpression profiling in experimental silicosis ratsrdquo The Journalof Toxicological Sciences vol 37 pp 1207ndash1215 2012

[26] H-S Gao R Xue P Ding et al ldquoCross-talk of the relatedbioactivity mediators in serum after injection of soluble TNF-120572 receptor on silicosis model of ratsrdquo Toxicology and IndustrialHealth vol 27 no 7 pp 607ndash616 2011

[27] X Wang Y Chen L Lv and J Chen ldquoSilencing CD36 geneexpression results in the inhibition of latent-TGF-1205731 activationand suppression of silica-induced lung fibrosis in the ratrdquoRespiratory Research vol 10 article 36 2009

[28] X T Wang J Liu L H Liu and J Y Zhao ldquoAnalysis ofinfluencing factors on infrared spectroscopic determination ofthe content of free silica in quartz sandrdquo Zhonghua Lao DongWei Sheng Zhi Ye Bing Za Zhi vol 29 pp 622ndash625 2011

[29] Y He Q Miao and B Liu ldquoEffects of polyvinylpyridine oxideon gene expression of type I and III collagens in experimentalsilicosisrdquoChinese Pharmaceutical Journal vol 30 no 5 pp 271ndash273 1995

[30] Z P Chen W G Su and S Yan ldquoTherapeutic effect of Tetran-drine on patients with pneumoconiosisrdquo Journal of ClinicalPulmonary Medicine vol 17 pp 1226ndash1227 2012

[31] J Gao Z Zhang X Ji et al ldquoFurther discussion on the indica-tions and contraindications in whole-lung lavagerdquo Occupationand Health vol 27 pp 410ndash412 2011

[32] Z H Zhang H Liu andG XMa ldquoClinical analysis of the 5000cases of pneumoconiosis and other lung disorders treated bylarge volume whole lung lavage treatmentrdquo Chinese Journal ofConvalescent Medicine vol 10 pp 956ndash960 2009

[33] Q F Zhang Z Zhang G G Mao Q zhang and Y Du ldquoDataanalysis and experimental study on pathogenic therapy or lunglavage for dust removalrdquoOccupationalMedicine vol 27 pp 4ndash62000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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EndocrinologyInternational Journal of



Disease Markers


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


regulate blood pressure slow aging and prevent and treatvarious diseases [7] Kombucha contains two notable groupsof microorganisms Gluconoacetobacter xylinus (Xylinum)and yeasts Xylinum secretes bacterial cellulose through holesin its cell walls Interestingly kombucha cultures producebacterial cellulose more efficiently than cultures of Xylinumalone [8 9] The bacterial cellulose produced by kombuchacultures has a number of properties that make it potentiallyuseful inmedical applications including good biocompatibil-ity thinness (the cellulose sheets are typically only 01micronsthick) and a high specific surface areaThese propertiesmeanthat it functions as a nanoscale functional material with adefined three-dimensional structure and a large number ofsurface-exposed hydroxyl groups which allow it to formstrong noncovalent bonds with water and also a wide range ofions and organic compounds [10] It has been demonstratedthat bacterial cellulose efficiently adsorbs numerous toxicheavy metal ions including Cu2+ Pb2+ Hg2+ and Cd2+ Inaddition it adsorbs a range of nonmetallic toxins such ashistamines ammonia and NO2minus and formaldehyde [11ndash16]We therefore hypothesized that if kombucha were sprayedinto lung tissues the bacterial cellulose produced by theculturemight adsorb dust and protein precipitates that wouldotherwise cause the symptoms of silicosis

Chinese herbal kombucha preparations can be madeby fermenting extracts of various plants with a kombuchaculture If the plants used in these preparations containbiologically active substances the resulting Chinese herbalkombucha may combine the beneficial effects of the kom-bucha itself with those of the plants A range of plants andherbs can be used for this purpose including licorice Siratiagrosvenori mangosteen and chrysanthemum Licorice isknown to have numerous pharmacological effects and iswidely used in traditional medicine It contains a range ofbiologically active compounds including glycyrrhizin gly-cyrrhetinic acid and the licorice flavonoids Both licorice andthe isolated active compounds are known to have antitussiveand expectorant properties Moreover it is effective againstasthma and offers protection against respiratory pathogens[17 18] Siratia grosvenori is a specificChinese plant belongingto the family Cucurbitaceae When dried its fruit is verysweet and has a cooling effect It is an important ingredientof ldquocooling drinksrdquo and is considered to be beneficial forrelieving conditions characterized by high body tempera-tures such as inflammation It is used in traditional Chinesemedicine to treat chest pains dry coughs sore throats andaphonia [19] Mangosteen aqueous extracts have been shownto significantly reduce the frequency of coughing in guineapigs treatedwith citric acid or capsaicin and inhibits coughingcaused by mechanical stimulation [20] Chrysanthemumis an important medicinal plant that has various pharma-cological effects Among other things it functions as anantibacterial anti-inflammatory vasodilatory and antitumoragent as well as reducing blood pressure and acting as anantioxidant [21] Teas made from the buds of the camelliaplant containmany bioactive compounds including polyphe-nols tea polysaccharides alkaloids vitamins and aminoacids as well as various essential metal ions As a result theyhave a wide range of pharmacological activities including

anti-inflammatory antioxidant and antithrombosis effectsas well as being useful in the treatment of diabetes and forreducing blood pressure [22 23]

In light of these facts we sought to investigate the utilityof kombucha and various Chinese herbal kombuchamixturesfor the treatment of silicosis and other diseases arising fromthe presence of dust in the lungs To this end rats wereexposed to silica dust via tracheal injection and then sprayedwithChinese herbal andnon-Chinese herbal kombucha solu-tionsThe results of the kombucha treatments were comparedto those observed following treatment with tetrandrine acompound that is known to be useful for mitigating thesymptoms of silicosis [24] The different treatments wereevaluated in terms of their effects on lung anatomy collagenlevels in the lung tissues of the experimental animals andtoxicity

2 Materials and Methods

21 Materials Silica dust (99 particle diameter 05ndash10 120583mwith 80 of particles having diameters of 1ndash5 120583m) waspurchased from Sigma Aldrich Tetrandrine was purchasedfrom the Zhejiang Zhongyi Pharmaceutical Co Ltd A kit formeasuring collagen (hydroxyproline) levels was purchasedfrom the Nanjing Jiancheng Bioengineering Institute

22 Preparation of the Kombucha Mixtures Kombuchastrains were purchased from the Beijing Institute of FoodResearch

A kombucha stock solution was prepared according tothe method of Ai-jun Lv [23]The Chinese herbal extract wasprepared bymixing tea (02ww) licorice (05ww) driedSiratia grosvenori fruit (05 ww) and wild chrysanthemum(02 ww) in water and boiling the resulting mixtures for20minThe boiled solution was then filtered cooled to below30∘C and mixed with a 20 dilution of the kombucha stocksolution After fermenting at 30∘C for 2 weeks the Chineseherbal kombucha solution was considered ready for use inexperiments

23 Experimental Animals Test animals were provided bythe Hunan Slack Jingda experimental animal company(License number SCXK Hunan) The animals used wereSpecific Pathogen Free (SPF) Sprague-Dawley (SD) rats thathad been held under quarantine for 5 days

24 Tracheal Injection of Method for the Contamination ofSilica Dust [25ndash27] A 50mgmL standard suspension ofquartz (silica) dust in saline was prepared Prior to trachealinjection samples of the stock solution were autoclaved andthen mixed with mycillin (4000UmL) The experimentalanimals were then anaesthetized under sterile conditions andsubjected to intratracheal injection with 1mL of the sterilemycillin-containing silica dust suspension in lungs

25 Experimental Groups and Treatments The 150 experi-mental animals weighed 180ndash220 g each and were randomly



Group Treatment















4 Results












2224 plusmn 179998779

2381 plusmn 220 2498 plusmn 290




2208 plusmn 190998779

2372 plusmn 236 2484 plusmn 267















































6 045 plusmn 018 073 plusmn 022998779


998779



998779














(mg)












5 Discussion






(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Group Treatment















4 Results












2224 plusmn 179998779

2381 plusmn 220 2498 plusmn 290




2208 plusmn 190998779

2372 plusmn 236 2484 plusmn 267















































6 045 plusmn 018 073 plusmn 022998779


998779



998779














(mg)












5 Discussion






(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2224 plusmn 179998779

2381 plusmn 220 2498 plusmn 290




2208 plusmn 190998779

2372 plusmn 236 2484 plusmn 267















































6 045 plusmn 018 073 plusmn 022998779


998779



998779














(mg)












5 Discussion






(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































6 045 plusmn 018 073 plusmn 022998779


998779



998779














(mg)












5 Discussion






(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























(mg)












5 Discussion






(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e)












Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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