Year  1997
Author  Igor  Afanas’ev,Natalia Polozova, Anatolii Dorozhko, Elena Ostrachovich





Technological regulations




Approved :

Professor Vladimir Gunar
Doctor of Chemical Science
Director General of Vitamin Research Institute



MOSCOW,  1997








Professor Igor  Afanas’ev
Head of Laboratory, Ph.D., D.Sci.


Research  workers:

Natalia Polozova
Senior Research worker, Ph.D. 

Anatolii Dorozhko
Senior Research worker, Ph.D. 

Elena Ostrachovich
Senior Research worker, Ph.D.





BN Soluble Application Guideline


Solution 5%
Preparation Sterilizer (autoclave) solution at over 200°C (possibly 250°C or above) or expose to γ- irradiation or ultrasound challenge.
Infusion 20 drops/min. Maximum 200 mL per infusion
Test Check blood sugar and others by endocrinologist at least 2 days after the cessation of the infusion.
Notes: Blood sugar level will go up. Watch carefully. After infusion, there may be fever. Observe carefully 2nd, especially after 3rd infusion when there may be immunological reactions.)



Process of BN Soluble




↓    →  → Remove unsoluble  matters

BN aqueous solution

↓     ←←   Aceton


↓       Centrifugation → → Fraction I

Soluble BN

↓                          Vacuum dry (30°C)

Fraction II



Principal parameter

Melting point:130-l35°C
pH value of 20% aqueous solution:  4.5 ~ 5.0
Specific rotation:+59.5 ~ 61.0° (1% aqueous solution)
Specific biological activity:Inhibition of luminal-amplified CL produced by zymosan-stimulated rat peritoneal macrophages, I50=8-8.5mg/mL
Solubility: Soluble in water but not in organic solvent
Storage: Stable at least 12 months under no exposure to water and light


Glucose  Biological activity (higher number stronger)
BN 90-92% 1
Fraction I 85-87 2
Fraction II 80-85 3





CHAPTER 1      Properties of final product

CHAPTER 2      Chemical scheme of the process

CHAPTER 3       Technological schemes of the process

CHAPTER 4       Raw materials, reagents and products

CHAPTER 5   Equipment

CHAPTER 6      Description of technological process

CHAPTER 7       Information data








1.1 Name:            Soluble Bio-normalizer (Fraction I)


1.2 Origin:       The product is prepared on the basis of Bio-normalizer, a natural Japanese food supplement prepared by the fermentation of Carica papaya (Sun-O International Inc., Gifu, Japan).

1.3 Biological activity: The product is nontoxic substance possessing antioxidant and immunostimulatory activities. It’s supposed area of application is the treatment of patients with  diabetes  mellitis, hepatitis,  and  various  inflammatory diseases  as  well  as  people exposed to environmental and therapeutic irradiation.

1.4      Principal parameters.


1.4.1   Product is a white or yellowish powder.


1.4.2   Melting point   130-135°C.


1.4.3   pH value of 20% aqueous solution    4.5-5.0


1.4.4   Specific rotation +59.5-61.0° (1% aqueous solution)


1.4.5   Specific biological activity:              Inhibition of luminol-amplified CL produced   by zymosan-stimulated rat peritoneal macrophages, 150 = 8-8.5 mg/mL.


1.4.6   Product is completely soluble in water and insoluble in organic solvents.


1.4.7   Product is stable during 12 months at least. It should be stored under conditions excluding exposure to moisture and light.








BN      water, 20°C →Soluble component + Insoluble sediment


Soluble component      acetone/water → Soluble BN (Fr.I) + Fr.II


Soluble BN (Fr.I) is precipitated after the addition of acetone to aqueous solution.
Fr.II is the second active product obtained by the evaporation of acetone-water solution after the separation of Fr.I.








Dissolution of BN

in water

BN suspension

1 centrifugation ——————————————————–> Insoluble sediment

Aqueous BN solution

↓ +acetone

Precipitation of Fr.I

2 centrifugation ————————-> Water/acetone solution of Fr.II

↓ rinsing with acetone                         ↓

↓                                                                        ↓

wet Fr.I                                                       Separation of Fr.II by distillation

↓ drying                                                           ↓

↓                                                                                    ↓

Final product: Soluble BN (Fr.I)                     wet  Fr.II

↓ drying                                                                                                                                                           ↓

Final product (Fr.II)











Bio-normalizer (BN), a natural Japanese food supplement prepared by the fermentation of

Carica papaya (Sun-O International Inc., Gifu, Japan).


Acetone produced in Russia (TU 6-09-3513-86; OSCH, 99.5% purity).

Distilled water.





(recommended for manufacturing Soluble BN on a factory)

N*) Name Number Material Parameters
1 Container for BN dissolution 1 Stainless steel Vertical bulk vessel with a jacket and a stirrer
3 Centrifuge 1 Stainless steel 13500 revolution/min
6 Container for precipitation of Fr.I 1 Stainless steel or enamel Vertical bulk vessel with a stirrer
9 Centrifuge 1 Stainless steel Centrifuge with a filter,1500 revolution/min
11 Apparatus for evaporation of  acetone 1 Stainless steel Vessel supplied with a rotor for dispersing the solution in vacuum
16 Rectification column 1 Stainless steel
12 Container 1 Stainless steel Vertical bulk vessel
8 Condenser 1 Stainless steel or enamel Vertical vessel with pipes
13 Condenser 1 Stainless steel Vertical vessel with pipes
17 Condenser 1 Stainless steel or enamel Vessel with pipes
19 Condenser 1 Stainless steel Vertical vessel with pipes
15 Trap 1 Stainless steel or enamel Vacuum-tight vertical vessel with a cooling jacket
4 Container 1 Stainless steel Vertical vessel
10,14,18 Storage tank 3 Enameled cast iron Vertical vessel
20 Storage tank 1 Stainless steel Vertical vessel
2,7 Measuring container 3 Stainless steel Vertical vessel

*) See, the scheme of equipment.







1. Preparation of BN suspension

BN (167.5 g) and distilled water (420 mL) were placed in a glass vessel with a condenser and a stirrer and were stirred for 20 min at 20°C (Table 6.1).


Table 6.1

N Product Loaded
1 BN 167.5
2 Distilled water 420
3 BN suspension 587.5 530




2. First centrifugation

BN suspension (530 mL) was centrifuged at 10000 g for 15 min (Table 6.2)


Table 6.2

N Product Loaded
1 BN suspension 587.5 530
2 Aqueous solution of Frs. I and II 577.3 520
3 Insoluble sediment 10.2
4 Total 587.5 587.5


3, Precipitation of Fr. I


Aqueous solution of Fr. I and II was placed the glass vessel with a condenser, a stirrer and a separator. After that, an 7.5 excess of acetone was added during 30 min at the continuous stirring of reaction mixture. The sediment (Fr. I) was separated by centrifugation (5000 g, 10 min).

Table 6.3

N Product Loaded
1 Aqueous solution of Frs.I and II 577.3 520
2 Acetone 3322.3 3900
3 Wet Fr.I (sediment) 131
4 Acetone/water solution of Fr.II 3764.8 4610
5 Losses (Fr.I) 3.8
6 Total 3899.6 3899.6


4.  Drying of Fr. I in vacuum.

Wet Fr. I was dried in vacuum vessel at room temperature for 15 hours (Table 6.4).
Average yield of Soluble BN (Fr. I) is 73.4%.

Table 6.4

N Product Loaded
1 Wet Fr .I 131
2 Dried Fr. I 123
3 Losses Fr. I Acetone 0.6
4 Total 131 131


5. Separation of Fr.II by distillation of acetone/water mixture.

Acetone and water were vacuum-distilled from the acetone/water solution of Fr. II (150 -15 mm Hg) at 35°C, and the wet Fr.II obtained was dried in vacuum vessel at room temperature for 20 hours (Table 6.5). Average) yield of Fr. II is 19.5%.


Table 6.5

N Product Loaded
1 Acetone/water solution of Fr.II 3764.8 4610    
2 Dried Fr.II     32.4  
3 Acetone/water
    3347.5 4080
4 Losses
5 Total 3764.8   3764.8  







Present documentation is the technological regulations of a principally new process of manufacturing the water-soluble fraction (Fr.I) of Bio-normalizer (BN), a natural Japanese food supplement prepared by the fermentation of Carica  papaya (Sun-O International Inc., Gifu, Japan). Water-soluble Fr. I possesses all the biochemical activities of BN and, to a certain extent, surpasses it. As a side product, the second fraction (Fr.II) was obtained, which exhibits even greater activities, but the production of this product is not a subject of present documentation.

The developed method of the production of Fr.I includes the following stages:

1. Dissolution of BN in water.

2. Centrifugation of BN suspension and the separation of insoluble sediment.

3. Precipitation by acetone and the separation of Fr. I by centrifugation.

4. Drying of Fr.I.


7.1 Choice of optimal conditions of BN dissolution.

To find optimal conditions of BN dissolution in water, a dependence of the yield of insoluble sediment on temperature and duration of the experiment as well as the amount of water has been studied (Table 7.1).


7.2 Choice of optimal conditions of the sedimentation of Fr.I.

For the isolation of soluble BN fraction the method based on the sedimentation of this product by acetone was chosen.  We found that BN is completely insoluble in acetone; therefore, the addition of acetone to aqueous solution of BN ought to result in the sedimentation of water-soluble fraction of BN. It is also known that acetone, which is frequently used as a solvent in manufacturing of drugs and food supplements, does not react  with natural organic compounds  at room  temperature,  accessible as  a  very  pure substance, and can be completely removed during the drying stage. It should be noted that the use of other solvents such as ethanol instead of acetone resulted in a very low yield of Fr I (<10%).

It was also shown that an important factor is the sequence of the operations: acetone should be always added to BN solution and not vice versa. When the BN solution was added to acetone, the yield of Fr.I sharply decreased.  If needed, centrifugation can be substituted by the filtration of the sediment of Fr. I from solution. However, in this case the losses of  acetone  enhanced,  and  its  content  in  the sediment  increased  that  made  the subsequent stage of drying more difficult. A dependence of the yield of Fr.I on the amount of acetone added during sedimentation is shown in Table 7.2.


7.3 Optimal conditions of the whole process of the manufacturing Fr.I

On   the   grounds   of   the   above   experiments   the   following   optimal   conditions   of manufacturing Fr I were chosen:


The amount of water is 2.5 L/1 kg BN.

The duration of dissolution is 20 min.

The temperature of dissolution is 20°C.

Insoluble sediment is cetrifugated at 10000 g.

Fr.I is cetrifugated at 5000 g.

The ratio of acetone/EN solution is 7.5:1

Fr.I is dried in vacuum at room temperature for 20 hours.


To show the reproduction of these conditions, a series of 5 experiments was carried out (Table 7.3). It is seen  that  the  average yield  of  soluble  BN  (Fraction  I) is  equal  to (73.4±0.6l)%


Table 7.1

N of


Amount of water/

1 g BN (mL)





Amount  of sediment(%)


1 1.0 20 20 1.05
2 2.0 20 20 2.35
3 3.0 20 20 2.45
4 4.0 20 20 2.44
5 2.5 16 20 1.80
6 2.5 20 20 2.45
7 2.5 25 20 2.47
8 2.5 30 20 2.45
9 2.5 20 10 1.2
10 2.5 20 15 2.1
11 2.5 20 20 2.45
12 2.5 20 25 2.45



Table 7.2

Dependence of the yield of Fr. I on the ratio of  acetone/aqueous BN solution.

N of exp. Ratio of acetone/aqueous
BN solution (v/v)
Yield of Fr.I
1 3.5
2 5.0 10
3 6.5 55.3
4 7.5 73.4
5 9.0 73.5



Table 7.3
Preparation of Fr. I under optimal conditions

N Temp of dissolution
Duration of dissolution
1 20 20 167.0 417 3900 4.08 529 4550 122 73.4
2 20 20 167.5 420 3900 4.15 520 4610 122 72.8
3 20 20 168.3 420 4000 4.05 525 4630 124 73.7
4 20 20 165.5 415 3800 4.13 515 4580 122.5 74..0
5 20 20 168.5 425 3900 4.05 520 4630 123.5 73.3
Mean 20 20 167.4±0.7 420±5 3900±100 4.10±0.04 520±7 4610±60 123±1 73.4±0.6







TLC analyses of BN powder, solubles BN (Fr. I), and soluble Fr. II were carried out on the Kieselgel 60 adsorbent in two solevents: solvent A (isopropanol/water, 4:1) and solvent B (normal propanol/ethyl acetate/water, 7:1:2).  Samples were applied as 1% solutions. Values of retention times (Rf) for BN samples were compared with Rr values for glucose, mannose, rhamnose, saccharose, galactose, and fructose (Table). It was found that the predominant component of BN powder, soluble BN, and Fr. II is glucose.




Sample Rin solvent A Rf  in solvent B
BN (powder) 0.62 0.50
Soluble BN (Fr.I) 0.62 0.50
Fr. II 0.62 0.50
Glucose 0.62 0.50
Mannose 0.65 0.55
Rhamnose 0.73 0.70
Saccharose 0.58 0.43
Galactose 0.54 0.42
Fructose 0.58 0.48




HPLC analysis is one of the most up-to-date sophisticated analytical methods, which permits to determine the quantitative composition of complex biological mixtures. For analyses of BN samples we used a Knauer chromatograph (Germany) with refractometric detector produced by Kova co. (Czech Republic).


Experimental conditions:

Column of 3.3 x 300 mm; modified silica gel Separon SQX-NH2 (an adsorbent); acetonitrile/water (8:2 and 1:1) mixtures as solvents; the fluid rate of 1 mL/min.  Samples were applied as 1% solutions.

The results obtained are given in Figures 1-3. It was found that in accord with the data of TLC analysis, the predominant component of all samples was glucose. Its retention time (8-8.033 min) completely coincided with that of glucose (8.033  min) and clearly differed from the retention  times of  mannose  (7.533  min),  rhamnose  (4.767  min),  saccharose (11.933 min), galactose (8. 80 min), and fructose (6.967 min). The content of glucose was estimated as 90-92% for BN powder, 85-87% for soluble BN (Fr. I), and 80-85% for Fr. II. As no other peaks were observed on the chromatogram even at the highest level of the chromatograph sensitivity (Fig. 4), we concluded that the other compounds containing in the samples (10-15%) apparently are not carbohydrates (sugars) because the compounds of different structure cannot be analyzed at the same conditions as carbohydrates.


or21_02 or21_03 or21_04 or21_05 or21_06






Optical and IR spectra were recorded for BN powder, the soluble BN (Fr. I), and Fr. II on a Specord 40 spectrophotometer (Germany) (Figure 1-3). Optical spectra were recorded for aqueous solutions, and IR spectra were obtained in a KBr cuvette. It is seen that all samples absorbed light at wave length λ > 350 nm. It is of interest that the spectrum of the soluble BN contained 3 maxima at 256, 275, and 290 nm (Fig.2), which were absent in the spectra of BN powder and Fr. II. This fact probably indicates the presence some unknown active compounds in the sample of soluble BN.

As one may expect, IR spectra (Figs. 4-9) of all BN samples are similar to that of glucose (Figs. 10, 11). However, the spectra of soluble BN (Figs. 6, 7) and Fr. II (Figs. 8, 9) contain several additional bands at 520 (doublet), 1660 and 2980 (doublet) cm-1 that points out at the presence some unsaturated and nitrogen-contained compounds.




Chromatographic and spectral analyses of BN powder, the soluble BN (Fr. I), and Fr. II show that the principal component of all BN samples is glucose. The glucose content decreases from BN powder (90-92%>) to the soluble BN (85-87%), to Fr. II (80-85%). Chromatographic analysis supposes that the other components of BN samples are not carbohydrates (sugars). Spectral analysis indicates the possibility of the presence in the samples of unsaturated and nitrogen-containing compounds.


or21_07 or21_08or21_09 or21_10or21_11 or21_12 or21_13 or21_14or21_15 or21_16 or21_17








Male Wistar rats weighting 150 to 200 g were maintained under standard laboratory conditions, with chow diet and water ad libitum.


Ex vivo studies.


The  rats  were  divided  into four  groups:  group  1 (control 6  animals)  received  the intraperitoneal  injection of 1.0 mL sterile physiological saline; group 2 (6 animals) received the intraperitoneal  injection of 10 mg BN powder in 1.0 mL solution;  group  3 (7 animal ) received the intraperitoneal  injection of 10 mg soluble BN (Fr. I) in 1.0 mL solution; group 4 (6 animals) received  the intraperitoneal injection  of 10 mg Fr. II in 1.0 mL solution.  24 hours later animals were sacrificed under slight ethyl ether narcosis.


Isolation of peritoneal macrophages.

Peritoneal macrophages were prepared by peritoneal lavage with 2 mL prewanned saline solution. The lavage fluid was filtered and centrifuged at 300 x g for 10 min. Cells were resuspended, twice washed, and stored at 4°C in I-JESS. Macrophage preparations were > 90% pure and > 95% of the cells excluded trypan blue.


Chemiluminescence assays

CL measurements  in a Luminometer mod  1251  ( LKB,  Sweden) were monitored  at 37°C and  continuous  mixing on  a  programmed  IBM  computer.   All experiments   were carried out in dublicate.  Each point was a mean of 3 or 4 independent measurements. Lucigenin-amplified  CL  was  applied  as a  test for  measuring  superoxide  production  and luminal-amplified CL was used as a test on active oxygen species forming during the decomposition  of hydrogen  peroxide, principally, hydroxyl radicals.


Measurement of lucigenin- and luminal-amplified CL produced by rat peritoneal macrophages.  Macrophage suspension (5.0 x 105 cells) and luminal (20 µM) or lucigenin (400 µM) were incubated in HBSS (pH 7.4, a total volume of 1 mL) in the CL unit of a lurninometer for 3 min. After measurement of background for 5 min, CL was activated by adding 0.5 mL (1 mg/mL)   opsonized   zymosan suspension.   An amplitude  of  the  CL response   to  an  activator  was  defined  as  the  difference   between  maximal  intensity  of activated CL and intensity of spontaneous CL.


In vitro studies

Experiments with peritoneal macrophages.

Lucigenin-   and   luminol-amplified  CL   produced  by  rat   peritoneal  macrophages   was measured  as in the ex vivo experiments  after adding  the appropriate  concentrations  of BN powder,   the  soluble  BN,  and  Fr.  II to the incubation   mixture containing  macrophage suspension.

Experiments with xanthine oxidase.

Lucigenin   (500   µM), xanthine   oxidase   (30   U),   and   a   BN   sample   of   appropriate concentration were incubated in phosphate buffer (pH 7.6).. The reaction was started by the addition of 100 µM xanthine.  After that, the CL intensity was measured.



All results are from the experiments carried out in dublicate or triplicates are presented as mean± SD.  Differences were analyzed using the Student’s t-test, the level of significance being set at P < 0.05. Each point was a mean of three independent measurements.





It has earlier been shown by us [ J. A. Osato,  I. B. Afanas’ev, Z. P. Cheremisina, T. B. Suslova, N. E. Abramova, E. V. Michalchik, I. B. Deeva, L. A. Santiago, L. G. Korkina, Phys. Chem. Biol. Med. 2,  87-95  (1995);  J.A.Osato,  L.G.Korkina,  E.  Michalchik, I.B.Afanas’ev, in Proceedings of the International Symposium on Natural Antioxidants: Molecular Mechanisms and Health Effects, L.Packer, et al., Eds., AOCS Press, 1996, pp. 109-116]  that the stimulatory activity of  Bio-normalizer can be  measured  by  luminol­ amplified CL in the ex vivo  experiments and its antioxidant activity can be determined in the in vitro experiments. Present data fully confirm this conclusion. As is seen from Fig. 1, the injection of rats with BN powder, soluble BN (Fr. I), and Fr. II led to the drastic enhancement of luminal-amplified CL by zymosan-activated macrophages. As the main component of BN samples is glucose, we compared the effects of all BN samples with that of glucose. It is seen that the effects of all BN preparations were significantly higher that the effect of glucose, but an especially great difference is observed for soluble fractions: thus, solubles BN (Fr. I) and Fr. II enhanced CL produced by zymosan-stimulated macrophages by 2.2-2.8  times in comparison  with BN  powder and  by 3.4 – 4.4  times in comparison with glucose. It follows that the stimulatory activity of  all BN  preparations cannot  be explaned  by the  presence  of  glucose  and  that  the activities of  soluble  BN fractions are significantly higher than the activity of the parent BN powder.

Antioxidant activities of BN preparations were determined in the in vitro experiments.

1, 2, 4, or 10 mg/mL of BN samples were added to the incubation mixture containing macrophage suspension (5.0x 105 cells) and luminal (20 µM) or lucigenin (400 µM) in HBSS (pH 7.4, a total volume of 1 mL). The reaction was activated by 0.5 mL (1 mg/mL) opsonized zymosan suspension.  As is seen from Figs.  2 and 3, all BN preparations suppressed the formation of active oxygen species measured both luminol- and lucigenin­ amplified CL in a concentration-dependent manner, while glucose had no effect or even enhanced free radical formation.  Antioxidant activity of soluble BN was close to that of BN powder, while Fr. II suppressed the formation of active oxygen species by 10-20 times more efficiently than BN powder or soluble BN (Fr. I).

In vitro antioxidant activities of BN preparations were also measured in the xanthine­ xanthine oxidase system, which is a well-known classic enzymatic systems widely used for the generation of superoxide ion. The inhibitory effects of BN samples and glucose on lucigenin-amplified CL produced by xanthine oxidase are shown in Table 1. It is seen that all 4 substances inhibited superoxide generation in this enzymatic systems, Soluble BN (Fr. I) and Fr. II being again the most effective scavengers of superoxide ion.


Table 1

The  effects  of  BN  preparations  and  glucose  on  lucigenin-amplified CL  produced  by xanhtine-xanthine oxidase system

Cl intensity
Control 134±7
Glucose 108±3
BN powder 111±7
Soluble 100±2
Fr. II 78±1







Fig. 1.        Lurninol-ampli:fied CL by zymosan-stimulated rat macrophages
(Ex vivo experiments).



Fig. 2. Luminol amplified CL by zymosan stirnulated rat peritoneal macrophages
(In vitro experiments).



Fig.3. Lucigenin-amplified CL by zymosan-stimulated rat peritoneal macrophages
(In vitro experiments).





1. The developed process of the preparation of soluble Bio-normalizer consists of 5 stages. All stages are the up-to-date technological processes, which were reproduced on a pilot plant. Prepared documentation corresponds to the conditions needed for the organization of manufacturing the soluble BN on the factory.


2. Both isolated soluble BN fractions I and II not only retain the immunostimulatory and antioxidant activities of parent BN but exceed it by several times. It may be suggested that the source of this difference is the inhibitory effect of insoluble particles in BN powder.


3. Soluble BN was prepared with a high average yield equal to (73.4±0.6)%.  Taking into account  that  the biological activity of  soluble  BN  exceeds  that  of  BN  powder  by 1.5-2 times, the developed  process  allows to  receive  the final  product  (soluble  BN)  with 100-150%  yield  (in  the  units  of  its  activity).  This fact makes the developed technological process a highly effective one for manufacturing IV BN preparations.


4.  On  the grounds  of  biochemical studies, it is concluded  that  the  soluble  BN  fractions differ  from  BN  powder  only  by the  enhancement of  their activities  but retain  the same biochemical properties. It justifies their application for the treatment of the same illnesses as parent BN.


5.  The enhanced biological activities of soluble Fractions I and II are a sound basis for the further investigation of their biochemical and pharmacological properties that will certainly enlarge the medical fields of Bio-normalizer application.