GROWTH ENHANCEMENT OF SCHIZOSACCHAROMYCES CERVEVISIAE WITH HEARBS EXTRACTS TO PREPARE A POTENT KAMBUCHA (TEA) FORMULA THAT CORRECTED LIVER PATIENT DISEASES OF MALE & FEMALE HUMANS STUDY IN THERAPEUTIC NUTRITION.

Abd El- Rahman Attia*;  Khaled Shaheen** , Tarek Abd El-Rahman**and Ayat El-Hossany**

* Department of Nutrition and Food Sciences , Faculty of Home Economics, Helwan University, Egypt

** Department of Nutrition and Food Sciences Faculty of Home Economics, Minufiya University,  Egypt

ABSTRACT

 To enhance the growth of Schizosaccharomyces cervevisiae the principal microorganism  Kambucha a total number of 14 herbs were used with the black tea .Herbs with vary thick growths (3herb) were excluded and the remained herbs (Ammi visnage,  Cassia Occidentalis,, Cichorium Inty bus, Erythraea Centaurium, Hibiscus sabdoriffa sabdariffa, Hordeum vulgare, Matricaria chamomilla, Rheum Rhoponticum , salvia officinalis , thymus vulgaris, Rosmarinus officinalis ) were used to purpose 6 groups of herb blends .such blends on (with a control with only tea )were is used, and only 3 of them were selected based on the most proper growth of Kambucha pellicle. The liquid (tea) of these three groups were mixed at the ratio (1:1:1 v/v/v) and the final liquid (called also Kambucha tea) was given to liver patients (1cup) with a cup of warm water and one tablespoon of bee honey for 3 months & on this steps (1,2&3 times a day in the 1^st,2^nd and 3^rd  month respectively) Liver out patients of present study (males & Females) were 10-63 years old and the trial was with participation of a doctor (consultant of liver and immunity).Food habits revealed good practices such as like fresh vegetables & fruits and preferring boiling for cooking; or and other not correct as 31.9% of females and 9.0% of females drink only 500 ml of liquid per day .Out patients had also other disease ( besides liver disease) such as hypertension, diabetes ,gout ,gravel, anemia osteoporosis , renal failure , and others. They suffer of different kinds of liver disease  (fatty liver fibrosis, liver cancer, HAV, HBV, MCV, HBV+HCV and hypertrophy +spleenomegaly);also parasitic infection, nasal hemorrhage ,ascites ,trema and dizziness were found .Suggested treatment of present work corrected the level of GPT,GOT,ALB ,BIL ,ALK.P & T. prot. to nearly the normal level ,indicating the value of this remedy.

Key words:  Liver patients, Kambucha tea, herbs extracts, serum analysis.

INTRODUCTION

A lot of statistical healthy facts that investigated obviously push towards vigorous work in this research. Background The population of Egypt has a heavy burden of liver disease, mostly due to chronic infection with hepatitis C virus (HCV). Overall prevalence of antibody to HCV in the general population is around 15-20%. The risk factor for HCV transmission that specifically sets Egypt apart from other countries is a personal history of parenteral antischistosomal therapy (PAT). A review of the Egyptian PAT mass-treatment campaigns, discontinued only in the 1980s, show a very high potential for transmission of blood-borne pathogens. The relative importance of PAT in the spread of HCV in the Egypt was studied (WHO, 2006) and may be displayed as follows:

Methods of the degree of exposure to PAT by cohort were estimated from 1961-86 Ministry of Health data. A cohort-specific exposure index for PAT was calculated and compared with cohort-specific HCV prevalence rates in four regions.

Findings HCV prevalence was calculated for 8499 Egyptians aged 10-50 years. A significant association between seroprevalence of antibodies to HCV and the exposure index (P=0.007) was identified across four different regions. In all regions cohort-specific HCV prevalence was lowest in children and young adults than in older cohorts. These lower prevalence rates coincided with the gradual and final replacement of PAT with oral antischistosomal drugs at different points in the time in the four regions (WHO, 2006).

Interpretation the data suggests that PAT had a major role in the spread of HCV throughout Egypt. This intensive transmission established a large reservoir of chronic HCV throughout Egypt. Also, this intensive transmission established a large reservoir of chronic HCV infection, responsible for the high prevalence of HCV infection and current high rates of transmission. Egypt's mass campaigns of PAT may represent the world's largest iatrogenic transmission of blood-borne pathogens. Egypt has a very high prevalence of HCV and a high morbidity and mortality from chronic liver disease, cirrhosis, and hepatocellular carcinoma. Approximately 20% of Egyptian blood donors are anti-HCV positive. Egypt has higher rates of HCV than neighboring countries as well as other countries in the world with comparable socioeconomic conditions and hygienic standards for invasive medical, dental, or paramedical procedures. The strong homogeneity of HCV subtypes found in Egypt (mostly 4a) suggests an epidemic spread of HCV. Since a history of injection treatment has been implicated as a risk factor for HCV, a prime candidate to explain the high prevalence of HCV in Egypt is the past practice of parenteral therapy for schistosomiasis. The large reservoir of chronic HCV infection established in the course of these campaigns remains likely to be responsible for the high prevalence of HCV morbidity and may be  largely responsible for the continued endemic transmission of HCV in Egypt today(WHO, 2006).

The present work was conducted to investigate the possibility of using Kambucha tea as remedy for hepatitis. Enhancement the bioactivity of Schizosaccharomyces cervevisiae (effecting Kambucha microorganism) was studied via selection among 4 herbs and inclusion of bee honey in the final suggested formula.

Subjects AND METHODS

1- Subjects:

This study was carried on a sample of patients (males and females), which are suffering from liver disease. The sample was selected randomly from outpatient department (of a hospital in Cairo) and consisted of 65 from males and females classified to (32 males and 33 females). The age of sample ranged between 10 to 65 years.

2- Methods:

2.1 Data collected from each patient include:

2.1.1. Food habits sheet and

2.1.2 Medical healthy sheet according to (Basiotis et al., 1987).

2.1.3. Height (HT),

2.1.4. Weight (WT) and

2.1.5. Body mass index (BMI) according (J’equier, 1987).

2.2. Statistical Analysis:

The data were analyzed statistically by using SPSS statistical program (SPSS, 1998).

2.3. Schizosaccharomyces cervevisiae with herbs.

2.3.1. Making Kambucha Tea according to (Anonymous, 2005).

3. Administration

The administration took place was on 3 steps gradually as the following:

1-    Table spoon with 1 cup of warm water +1 tablespoon bee honey for first3 days as the first step.

2-   Second period (after 3 days) 2 time a day.

3-   Third period after the second step, was 3 times/ day. 

            It should be mentioned that all 14 herbs were tested (Table A) and  3 of them were excluded (which gave very thin or very thick Kambucha pellicle) .The remained 11 herbs were used to prepare 6 groups of herb blend with a control with only black tea (table B) which were used to grow Kambucha. Three  groups only   were selected (last 3 groups in Table b) based on the most

3.2.2. Table (A): Reaction of Schizosaccharomyces cervevisiae as growth (positive or negative) for herb selection.

proper growth .Liquids of these there groups were mixed at the ratio (1:1:1 v/v/v) and this final liquid mixture (called final tea) was given to liver patient (1cup) with a cup of warm water and on tablespoon of bee honey for 3 months on three steps (1, 2 & 3 times a day in the 1^st,2^ndand 3^rd  month , respectively). 

Each selected herbal blend was prepared  as following , 2 cups of boiled tea (cooled to room temperature)+2 tablespoons sugar+ 1 cup of fermented liquid of Schizosaccharomyces cerevisiae + 1 cup of herb extracted in boiling water “cooled to room temperature prior to herb removing.

RESULTS

1-Food habits:

Data in Table 1 shows frequency distribution of liver disease patients (males and females) according to food habits. The results indicated that the highest percent was found among personas who take three males, it was 81.2% for males, while for females was 81.8%.In respect to skipped  meals, the highest percent was for patients who take all meals for males and females (84.4%, 81.8%) respectively. As for the higher (omitted none) percent of patients taking fluids it was (1500.00 ml / day) for males (25%) ,while the lowest (4000 ml/day) 31.2% of males were taking 500 ml/day. Meanwhile for females the higher of patients percent was 27.3% for persons, who take (500 ml / day) & 1500 ml / day).Nevertheless 36.3% of females was taking 1000 ml / day.In respect of taking milk, the percent for males and females was (78.1% & 100%) respectively.

2.3.3. Table (B):  Reactions of Schizosaccharomyces cerevisiae as  growth

                            (positive or negative) for herbs selection.

4. Laboratory analysis:

4.1  Determination of Got (AST): (Hafkenscheid, 1979).

4.2 Determination of GPT (A LT): (Anonymous  1980).

4.3 Determination of plasma alkaline phosphatase (Tietz , 1976).

4.4. Bilirubin: According to (Jendrassik et al., (1983).

The highest percent of drinking tea was for females (81.8%) while who don’t take tea highest percent for males was (37.5%).As for patients who take tea after meals the highest percent for males was (43.8%), while highest percent who don’t take tea after meals was for females (54.5%). Patients who take (light tea): The highest percent was for males and females, being 50.0% and 54.5% respectively. These results agree with that obtained by Sugiyama et al., (1999). Omitting tea and other beverages supress D-galactosamine-induced liver injury in rats. These authors  compared the effects of various types of beverages (tea, coffee, and cocoa) on D-galactosamine-induced liver injury by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferse (AST) activities in 7-wk-old male Wistar rats. The effects of five fractions extracted with different organic solvents from green tea, different types of dietary fibers, and some short chain fatty acids were also investigated. All of the beverages tested significantly suppressed D-galactosamine-induced enhancement of plasma enzyme activities when powdered beverages were added to the diet (30 g/kg) and fed to rats for 2 wk. Plasma ALT activities were 1155 +/- 82 [micromole/(min.L), control], 289 +/- 61 (green tea), 626 +/- 60 (roasted green tea), 471+/-84 (pure tea), 676 +/- 69 (oolong tea), 423 +/- 76 (black tea), 829 +/- 53 (coffee), and 885 +/- 89 (cocoa). The profile of AST activities was similar. The caffeine-containing fraction from green tea had no significant effect, whereas the other four fractions, including the soluble fiber fraction, significantly suppressed liver injury. In addition to tea fibers, many other types of dietary fiber (hemicellulose,  chitin,  chitosan,  alginate,  pectin,  guar gum,  glucomannan,

Table 1 : Frequency distribution of liver disease patients (males                   and females) according to food habits.

and inulin ( but not cellulose) had liver injury-preventive effects when added to the diet (30 g/kg) suggesting that liver injury-prevention may be one of the general effects of dietary fibers. Of three short-chain fatty acids tested (acetate, propionate, and butyrate), only acetate prevented liver injury when added to the diet (15 g/kg), supporting the possibility that the liver injury-preventive effect of dietary fibers may be mediated at least in part by certain organic acids. These results suggest that several beverages possess preventive effects on certain types of liver injury, such as that induced by D-galactosamine, and that different constituents of high and low molecular weights contribute to the liver injury-preventive effects of green tea.

Taking pickles. The highest of patients who don't take pickles was (for males and females) 62.5% and 72.7% respectively. For taking fresh vegetables the highest percent was for males and females, being of 93.7% and 100% respectively. Methods of cooking foods data revealed that for patients who take raw x raw vegetables the highest percent was (87.5% and 81.8%) for males and females respectively. As for methods used in cooking meat, chickens and fish, patients who use boiling at the highest percent for males and females were (56.2 % and 63.6%) respectively.

The highest percent of (males & females) patients who use small quantities of salt for males and females, the highest percent was (53.1% & 81.8%) respectively. Patients who use small quantities of spices for males and female at the highest percent were( 53.1% & 90.9%) respectively. All patients don’t take alcohol except 3.1% of males. Moreover all patients take vegetable and animal protein.

2-Other disease of liver patients:

Results in Table 2 showed the percent distribution of other diseases by questions and its answers and kinds of these diseases patients who suffer from another disease the highest percentage was in males and females 71.9% and 81.8 % respectively. The highest percentage was in patients who suffer from liver disease only 28.1% in males. While in another diseases like Gout, Gravel, Anemia companied with Electrolytes, Anemia companied with osteoporosis, psoriasis, Obesity companied with circular cholecystitis, piles, prostate and Anemia the percentages were equilibrium for males it were  3.1%.The highest percentage for females was 27.3%  had hypertension. While in another disease like Diabetes, digestive diseases, electrolytes, anemia added to osteoporosis obesity plus circular cholecystitis and renal failure the percentages were equivalent for females, it was 9.1%.

3-Kind of  liver disease.

Results in Table 3 show the percent distribution of kind for liver disease among patients (males and females). The highest percentage for males and females was HCV. It was 78.1% and 63.6% respectively for another diseases like fatty liver, fibrosis, liver cancer, HAV plus HBV the percentages were equivalent males. It was 3.1%. While in cases females another disease like fatty liver and Hypertrophy added to spleenomegaly. The percentages were same line values for females, it was 18.2%.

Data in Table 4 showed percent distribution of symptoms and parasitic infections among patient (males and females). The percentage of patients of liver Coma it was for males and females 15.6% and 9.1% respectively. While the percentage for patients of parasite disease were 18.8% and 9.1 % for males and females respectively. The percentage of patients of nasal hemorrhage was for males and females 50.0% and 36.4% respectively. About edema the percentage of patient's males and females was 53.1% and 81.8% respectively. While in asictes the percentage for males and females was 31.3 % and 36.4% respectively. In loss appetite the percentage for males and females was 15.6 % and 9.1% respectively. The percentage of patients of heavy perspiration was females and females 21.9% and 9.1% respectively while the percentage for patients of dizziness was 31.3% and 27.3% for males and females respectively about tremor the percentage for patients males and females was 15.6% and 27.3% respectively. As conclusions the total numbers were examined (32 males) vs. (33 females).

Table 2: Frequency    distribution    of     liver    patient's  (  males   and

               females) variable.

Table 3 : Percent distribution of kind for liver disease among patient

                (males and  females).

Table 4: Percent distribution of symptoms and parasitic infections among Patient (males and females).

Results in Table 5 showed mean ±SD and t- test values of anthropometrical measurements for  liver disease among patients (males and females) height (cm) mean± SD for males and females was (176.06 ± 11.361) and (164.90 ±6.5) respectively. Statistical data as t. test illustrated insignificant value (1.031) for males also, Height (cm) for females was insignificant (1.739) statistically data as t- test illustrated insignificant value (1.042) for males and females. Data was tabulated illustrated that weight (kg) as mean± SD for males (82.3428±15.9386) compared with females was (78.0±6.7676) the t. test for males was significant (1.743) (P<0.05). While females  values were very high significant (6.861) (P<0.001). Statistically t. test as compared with males and females were significant (P<0.05). Also, BMI (kg/m²) as mean ± SD for males and females was (26.49±4.3957) and (28.89±4.3051) respectively. While t. test for males was very high significant (-22.726) (P<0.001) and t. test for females was very high significant statistical t. test as compared with males and females were insignificant (0.001). 

Table 5: Mean ±SD and t- test values of Anthropometrical measurements for liver disease among patients (males and females).

* Significant P< 0.05, ** Highly significant P<0.01,  *** Very highly significant P<0.001.

Comparison between males and females of blood, serum analysis before and after herbal treatments:

Data was tabulated in Table (6-a & 6-b) and Figure 6. Obviously results of mean ± SD and t. test values of blood, serum analysis for patients (males & females) with liver disease before and after treatment. The GPT (/L) was (36.57±16.54) for males and was at normal states (normal was up to 40 u/l). t. test was (0.17) as a comparison and insignificant. Also values of GPT (u/l) for females at normal level was (36.09±22.04) and t. test was No significant t. test between males and females was (0.24) and insignificant. Not Got but also, GPT which formerly known as glutamate pyruvate transaminase was at normal level for both males and females it was (13.19 ±5.55 and 14.04±7.88) respectively. T. test for males and females was Non significant (0.55 and 0.46) respectively. T. test between males and females was 0.34 and insignificant.

      Table (6-a): Mean values of blood, serum analysis for patients (males

                            & females) with liver disease (Before treatment).

Table (6-b): Mean ±SD and t. test values of blood, serum analysis for patients (males & females) with liver disease (After Treatment).

* Significant P< 0.05,  ** Highly significant P<0.01,  *** Very highly significant P<0.001.

According cheesbrough, (1998). The enzymes Aspartate amino transeferase (AST), previously known as glutamate Oxalacetate transaminase (GOT) and Alanine aminotransferase (GPT), are concerned with amino acid metabolism. Large amount of AST are present in the liver, kidneys cardiac muscle small amount of the enzyme are present in the brain ALT is found principally in the liver with only small amounts being present in other organs. When there is liver cell damage the serum or plasma levels of both enzymes are raised.

The results were at normal levels that emphasis that the treatment with herbal and Schizosaccharomyces Cervevisiae would enhance viral hepatitis, the results agree with (Lin et al., 2003 & Jafri et al., 1999& Ibrahim et al., 2004 & Amin et al., 2005 & Emery et al., 1996).

ALB (g/dl) fore males and females compared with normal level was (4.67± 1.95, 4.97±2.79) and (3.8-4.4 g/dl) respectively and the results were at normal levels t- test was (0.38 and 0.38) for males and females respectively and was insignificant t- test between males and females was (0.29) and was insignificant. According (Strove, 1989) Albumin is produced entirely in the liver and constitutes about 60% of total serum protein. It is important in regulating the flour of water between the plasma and tissue fluid by its effect on plasma colloid osmotic pressure. When the concentration of albumin is significantly reduced the plasma osmotic pressure is insufficient to draw water from the tissue spaces back referred to as edema.

Albumin also has important binding and transport functions it binds and inactivates substances including calcium, Bilirubin fatty acid, urate, hormones and magnesium and also drugs when Alb-., level are reduced, toxic effects can develop from an increase in unbound substances. Serum albumin is mainly measured to investigate liver diseases .the results were normal that emphasis that the herbs Schizosaccharomyces Cervevisiae. Would enhanced liver and the results agree with (Broker et al., 1993 & Dragland et al., 2003 & Vitaglione et al., 2004 & Mroueh et al., 2004). Bilirubin (mg/dl) was determinate and results cleared values tend to be mild rise. The values for males and females were (0.60±0.25 and 0.64 and 0.36) respectively compared with normal level (up to 0.55 mg/ dl). t. test was insignificant (0.09 and 0.22) for males and females respectively. T. test among (males and females) was significant (0.05) (p<0.05).

According (Cheesbrough, 1998). The value of test measurement of serum or plasma Bilirubin is usually performed to investigate the causes of liver disease and jaundice and to monitor a patient's progress. The results investigated near to normal level and that emphasis that herbs and Schizosaccharomyces Cervevisiae enhance liver diseases the results agree with (Gilani et al., 1998 & Aktay et al., 2000 & Bornet et al., 2002 & Ahmed et al., 2003 & Amirghofran  et al., 2000).

AlK.P (1u/L) was determinate and results investigated that males and females achievement value to be mild raised (43.96±8.36 and 46.79±6.29) for males and females respectively. The t- test for males and females compared with normal level was very high significant (0.001 and 0.001) (P<0.001) respectively. The value of t- test compared with males and females was significant (0.03) (P<0.05) (Strove, 1989). Illustrated that (AP) Alkaline phosphatase used as indicator to liver disease the results tend to be mild raised and that emphasized that herb and Schizosaccharomyces Cervevisiae enhanced liver health the results agreed with (Makino et al., 2002 & Vilaglione et al., 2004 & Huang et al., 1997).

Total protein (g/dl) was determinate and data illustrated that both males and females at normal level (7.95±3.33 and 8.48±4.77), respectively. compared with normal level (4.6-7.15G/dl). Value of t. test between males and females was insignificant Also t. test between normal level and (males and females) was insignificant according cheesbrough, (1998). the total protein used as indicator to liver disease or gastrointestinal tract or loss protein from body as the result of disease the date was tabulated emphasis the herbs and Schizosaccharomyces Cervevisiae enhanced liver disease the results agree with (Pogna, 2002 & Gotta, 2005 and Miyagawa et al., 1997). 

Conclusively, the present study recommended treatment for liver diseases as consumed Kambucha tea as it described in present study for 3 months which corrected the level of GPT, GOT, ALB, BIL, and ALK.P & T. prot. To nearly the normal level, indicating the value of this remedy.

Acknowledgement

The authors of this name script are greatly indebted to Dr. Gooda Mohamed Awad ,Consultant of liver and immunity, Al-Azhar Hospital for his sincere help, support and valuable suggestion during this work.

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تشجیع نمو Schizosaccharomyces cerevisiae بواسطة مستخلصات الأعشاب لأعداد تولیفة شاى کمبوشا فعاله ﺇستخدمت فى علاج امراض الکبد لذکور وﺇناث اﻹنسان

*عبد الرحمن عطیه- **خالد علی شاهین-** طارق عبد الرحمن- **ایات الحسینى

*قسم التعذیة وعلوم الاطعمة - کلیة اﻹقتصاد المنزلى- جامعة حلوان.

** قسم التعذیة وعلوم الاطعمة کلیة اﻹقتصاد المنزلى- جامعة المنوفیه تغذیه.

            استخدم لتشجیع نمومیکروب السکارومیسیس سیرفیسیا Schizosaccharomyces cerevisiae عدد 14 عشبا مع کمبوشا الشاى الأسود.الأعشاب التى تنوعت فى ثخانة النمو(3أعشاب) قد استبعدت واستبقیت الأعشاب(بذور الخردل اى الخله البلدى، سنامکى، الهندباء، القنطریون، الکرکدیه، الشعیر، الشیح الأفرنجى، الرواند، المریمریه(الناعمه) السعتر، حصالبان) واسنخدمت بغرض اعداد 6 مجموعات من التولیفات العشبیه.وهکذا استخدمت مخلوطه على (مع الضابطه فقط مع الشاى)،وقد اختیر منهم ثلاثة على اساس النمو الأکثر ملائمه لقشرة الکمبوشا . وتم خلط السائل (الشاى) لهذة المجموعات الثلاثه بنسب(1:1:1 حجم) والسائل النهائى لهم( سمى شاى الکمبوشا) الذى اعطى لمرضى الکبد(1کوب) مع کوب من الماء الدافىء وملعقة مائده من عسل النحل لمدة ثلاثة اشهر و بالخطوات الآتیه(1،2 ثم 3 مرات / الیوم وذلک فى الشهور 1،2 ثم 3 على التوالى).

اجریت هذة الدراسه على مرضى الکبد من العیاده الخارجیه( ذکور واناث) فى عمر 10-63 عاما باشراک طبیب ( استشارى الکبد والمناعه). واوضحت العادات الغذائیه  تطبیقا جیدا کاشتمالها على  الخضروات و الفواکه الطازجه وتفضیل السلق عند االطهى ،بینما غیر الصحیحه منها 31.9% للذکورو 9.0% من اللأناث یشربون  500مل من سوائل فقط/الیوم. ومرضى العیادة الخارجیه یعانون من امراض اخرى      ( بجانب امراض الکبد) مثل  ارتفاع ضغط الدم، السکرى،الغده الدرقیه،الطفح الجلدى،انیمیا،هشاشة العظام،فشل کلوى وغیره ، ووجد انهم یعانون من انواع مختلفه من امراض الکبد( التدهن الکبدى،التلیف،سرطان الکبد،التهابات فیروسیه A,B,C,B+C، ضمور وتضخم فى الطحال) وایضا  العدوى بالطفیلیات، النزیف الأنفى، الأستسقاء،الرجفان و الدوار. فى هذة الدراسه کان التعامل على تصحیح مستویات GPT، GOT ، ALK.P، BIL و T.prot الى ما یقترب من المسنوى الطبیعى مما یدل على علاج واعد.

التوصیة: استخدام شاى الکمبوشا کما هو موضح بالدراسه الحالیه ولمدة 3 اشهرمما یصحح من قیم GPT، GOT ، ALK.P، BIL و T.prot الى ما یقترب من المسنوى الطبیعى مما یدل على علاج واعد.