Research article
Issue: № 8 (50), 2016

Гасанова Д.А.1, Гасанова З.З.2

1 Доцент, Кандидат медицинских наук, Кафедра фармакологии, Азербайджанский Медицинский Университет, Баку, Азербайджан, E-mail:, 2 Южно-Калифорнийский Университет, Лос Анжелес, Калифорния, США, E-mail:



У кроликов с токсическим повреждением печени тетрахлорметаном, было изучено фармакологическое действие  суммарного экстракта Фитокомпозиции №1+PhytoF (Ф№1+F) на фунциональное состояние гепатоцитов, а именно на метаболизм белков  в сравнении с классическим гепатопротектором Эссенциале форте Н (ЭфН) и экстрактом Paliurus spina christi (PSCh), входящего в состав (Ф№1+F). С этой целью был проведён полный анализ общего белка, альбумина, глобулинов, церулоплазмина (ЦП), входящего в состав α2-глобулиновой фракции, как одного из основных компонентов системы антиоксидантной защиты и адаптации организма к стрессовым факторам, а также был определён белковый коэффициент.

В результате исследований было установлено, что во всех исследуемых группах животных, фармакологическая активность (Ф№1+F) из всех изучаемых параметров метаболизма белков в наибольшей степени влияет на синтез (ЦП) и на количество глобулиновой фракции. Далее, в сравнительном аспекте, был определён коэффициент отклонения от контролей  (КОК) для (Ф№1+F), (ЭФН), (PSCh)по (ЦП)-ну, а также нами была определена гепатопротекторная активность (КГА), для исследуемых веществ, на основании данных по изменению белковых фракций.

Ключевые слова: метаболизм белков, церулоплазмин, токсическое повреждение печени, глобулиновая фракция, Фитокомпозиция№1+PhytoF.

Hasanova D.А.1, Gasanova Z.Z.2

1 ORCID -0000-0002-9626-8997, Assistant professor, Candidate of Medical Sciences, Department of Pharmacology, Azerbaijan Medical University, Baku, Azerbaijan,, 2 University of Southern California, Los Angeles, California,



The pharmacological effect of the total extract of Phyto composition №1 + PhytoF (F№1 + PhytoF) on the functional state of hepatocytes, namely the metabolism of proteins, in comparison with the classical hepatoprotectorEssentiale forte N (EFN) and extract of Paliurusspina-christi (PSCh), which is in the composition of (F№1 + PhytoF), was studied in rabbits with liver induced by carbon tetrachloride. To this end,the complete analysis of total protein, albumin, globulin, and ceruloplasmin (CP), which is in the composition of the α2-globulin fraction as one of the main components of the antioxidant defense system and the response of the organism to stress factors, was performedand the protein coefficient was determined. The research showed thatamong the investigated proteinsin allgroups of studied animals, the pharmacological activity of F№1+PhytoF had the greatest effect on the synthesis of CP and globulin fraction content. Further, in a comparative aspect, the coefficient of variation from controls (CV) for (F№1 + F), (EPN), (PSCh) was determined by (CP),and based on the data of the changes of the protein fractions, hepatoprotective activity of the studied compounds was established.

Keywords: protein metabilism, ceruloplasmin, toxic injury of liver, globulin fraction, Phyto composition+PhytoF.


Introduction.Various proteins, which are formed in some cells and utilized in others, constitute about 7% of blood plasma weight.Plasma proteins, the main of which is albumin, are synthesized by hepatocytes. The use of proteins (albumins, globulins, ceruloplasmin) occurs in different organs depending on their metabolic roles: in liver, kidneys, etc. Albumin is the only non-glycoprotein among plasma proteins, and it circulates in the blood for a long time.Violation of protein metabolism occurs in infectious hepatitis, in toxic liver damage, and if the liver injury is accompanied by malabsorption of intestinal amino acids, and in many other diseases.An indicator of the protein-synthesizing function of the liver is the concentration of certain plasma globulin proteins [2,14]. The significant decrease in total protein and albumin is observed in liver function failure. Total protein is the sum of all circulating blood proteins. Determination of the total protein is used in the diagnosis of liver diseases. Serum total protein in rabbits is in the range of 60.0- 83.0 g / l, α-globulins 12.1%. [3], [4].The increase in globulins is possible in pathologies of the liver parenchyma, in inflammatory processes of the connective tissue, cirrhosis of the liver, etc. [17].Ceruloplasmin (CP) is a copper containing plasma protein with 1046 amino acid residues [1] and it relates to the alpha-2 globulin fraction and has practical applications [16]. Albuminsconstitute the largest fraction of the blood plasma proteins. They maintain oncotic plasma pressure; they transport molecules and provide the reserve of amino acids. They bind and transport bilirubin. Hypoalbuminemia is observed with a decrease in the synthesis of albumin in the liver: hepatitis, cirrhosis [4,7]. Reducing albumin concentration in plasma is associated with a sharp restriction of its re-synthesis from amino acids in the liver [21].

Medicinal plants that are included in the total extract, "Phyto composition №1 + PhytoF” (F №1 + PhytoF) are used in various diseases. Thus,they are used in the treatment of diseases of the liver, jaundice [18, 19, 20], possess choleretic, anti-inflammatory actions [19], have antispasmodic effect and enhance the regeneration of damaged tissue [18]; carotenes and carboxylic acids in their composition play an important role in performing metabolism.

Artemizia absinthium, etc are included in the phytoand used as cholagoguefor the treatment of liver diseases [19]; they contain thujone as well as- thujoneand ketothujone. Thujoneaffects the central nervous system, has an antiseptic effect. It also includes plants that contain a prebiotic - inulin (IN),which is recycledby the microflora of the large intestine. IN strengthens the immune system and has a positive effect on liver function. It is effectively used for the treatment of hepatitis B and C in the complex therapy.

Sum of flavonoids from a number of studied natural cereals included in the (F №1 + PhytoF) exhibits an effective antioxidant (AO) activity. Very small doses in terms of rutin inhibit the chemiluminescence reaction (CL) by 30% and they have high antioxidant activity and also antiradical activity against radicals in the DPPH reaction [8].

Flavonoids (FL) are biologically active substances produced in plants that have antioxidant properties and activate enzymes in their organisms. There are over a hundred types of the FL, includingrutin, catechin (identified 12 types of catechins), (EGC-3-G), which are contained in the leaves of green tea (in the F №1 + PhytoF)and havea hundred times stronger antioxidant effect compared with vitamin C. Catechins - flavonoids are excellent antioxidants, polyphenols inhibit free radicals in the organism[22].

Pharmacological action of Essentiale forte N (EFN) is hepatoprotective. Essential phospholipids are the main elements of the cell membrane and cell organelles. Inliver diseases liver cell membranes and cellular elements are always damaged, which leads to functional, enzymatic disorders and a decreased ability to regenerate. Phospholipids of the drug (EFN) areendogenous phospholipids according to their chemical structures. Insertion of these high energy molecules in the damaged areas of the cell membranes of hepatocytes, restores the integrity of liver cells and promote their regeneration. This phospholipid structure of the cell membranes of hepatocytes "loosened", resulting in an increase of their strength and elasticity, and improvement of metabolism. Phospholipids of the preparation (EPN), are responsible for the metabolism of lipoproteins by transferring neutral fats and cholesterol to the sites of oxidation. It mainly occurs due to an increase in cholesterol binding ability of HDL.

Thus, metabolism of lipids, proteins and detoxification function of the liver are normalized. Cellular structure is restored and preserved.Connective tissue is not formed in the liver, which promotes natural regeneration of liver cells. Over 90% of ingested phospholipids absorbed in the small intestine. Polyunsaturated phosphatidylcholine enters blood with lymph flow and from there, mainly associated with HDL, to the liver.

Aim and tasks. Purpose of the study. To prove experimentally the advisability of the use of the total extract of plant origin, having the most pronounced hepatoprotective, antioxidant, antiradical, metabolic and immunomodulatory effects in toxic liver injury [8] as specialized factors to protect the body from the effect of toxic substances.

The tasks for achieving this goal are as follows:

1) To characterize the spectrum of pharmacologic effects of the Phyto composition on the severity of the correction of metabolic and functional disorders in experimental animal models of liver toxicity;

2) To compare the effect of the total extract (F№1 + PhytoF) with the drug (EFH) and extract of Paliurusspinachristi (PSCh) on the functional state of the protein metabolism and to determine the functionalsynthetic status of the liver in rabbits with liver failure induced by carbon tetrachloride;

3) To assess the antioxidant properties of the studied extracts from plants and pharmaceuticals (EPN) in the experiment on the model of carbon tetrachloride-induced liver damaged.

4) To compare pharmacological activities of the studied preparations: extracts (F№1 + PhytoF), (PSSh) and well-known hepatoprotector- (EFH), the functional state of protein synthesis, and antioxidant globulin, ceruloplasminin rabbits with liver failure induced by carbon tetrachloride.

Materials and Methods. Rabbits (weighing 2-2.5 kg.) were subjected to 10-day injections with carbon tetrachloride solution (CCL4). The tested compounds were administered per os in a volume of 5ml for the total extract (F№1 + PhytoF) and the extract of (PSCh). A solutionof (EPN) was administered intravenously at a dose of 165 mg / kg. The studies were conducted in 8 series: I series - intact animals treated with isotonic sodium chloride solution in the corresponding volume; II - with experimental toxic injury of the liver; III - treated within 10 days (F№1 + PhytoF) after the toxic liver injury induced by CCL4; IV –treated with (F№1 + PhytoF) within 20 days after toxic liver injury; V - for comparison drug (EPN) was introduced intravenously within 10 days after the induced toxic liver injury; VI - (EPN) was administered for 20 days, after the induced toxic liver injury; VII - extract from (PSCh), in the content of (F№1 + PhytoF) was introduced within 10 days after toxic liver injury induced by CCL4; VIII- extract (PSCh) was introducedwithin 20 days after toxic liver injury induced by CCL4.

In all series blood sampling was performedfor determination of total protein, albumin and globulin, ceruloplasmin after 10 and 20 days.

Determination of total serum protein by the biuret method using a set of "Human" company (Germany). The method is based on the principle that proteins form a blue-violet color in an alkaline medium with copper sulfate. To 1 ml. of the biuret 10 µl of serum reagent was added, and after 30 minutes absorbance was measured at the biochemical analyzer STATFAX –with 1 cm cuvette, at a wavelength of 540 nm.against control (biuret reagent).

To determine the level of albumin the reagent kit of the company "Human" (Germany) was used. The principle of operation is based on the interaction of albumin with bromocresol green with the formation of a blue compound. 1 ml bromocresol green was added into two tubes (test and standard), then 10 µlserum and 10 µl albumin standard solution (50g / l)were added,respectively, after 10 minutes it was measured at a biochemical analyzer STATFAX - in 1 cm cuvette at a wavelength of 540 nm against control.Ceruloplasmin (CP) was determined by the method of O.B. Siverin et al. [10]


The total protein content in rabbits treated with total phyto extraction (F№1 + PhytoF), increased by 12% (p ˂0.01) on the 10th day and 18% (p˂0.01) on the 20th day  of the treatment; albumin, respectively, by 36.7% and 51% at (p˂0.01); globulins - 0.96% and 0.98% at (p˂0.01); ceruloplasmin - 310% and 596% at (p˂0.01) compared with the control group.

The total protein content in the rabbits treated with the drug (EPN) was increased by 17.6% (p˂0.01) on the 10th day and 21.9% (p˂0.01) on the 20th day  of the treatment; albumin, respectively, increased by 39% and 57.3% at (p˂0.01); globulins - 1% and 0.95% at (p˂0.01); ceruloplasmin - 228% and 518% at (p˂0.01) compared with the control group.

The total protein content in the rabbits treated with the extract (PSCh), increased by 15.7% (p˂0,01) on the 10th day  and 11.6% (p˂0,01) on the 20th day of the treatment; albumin, respectively, by 41% and 30% (p˂0,01); globulins - 0.99% and 1% (p˂0,01); ceruloplasmin - 307% and 379% at (p˂0,01) compared with the control group.

To assess the pharmacological direction and the extent of changes in the parameters of the protein synthesis function of liver and CP, in the globulin fraction content in normal and pathological organisms and also after a course of treatment, we calculated the coefficient of variation from the controls (CV) using the formula:

CV = [(AMexp–AMint) / AMint] x 100%, where:

- AMexp - arithmetic mean of the parameter for animals in the experimental group;

- AMint - arithmetic mean of the parameter for intact animals.

Dynamics of CV of the synthesis function of liver for (F№1 + PhytoF) in the production of an important protein CP, belonging to α2-globulins, which is a powerful antioxidant, on 10th and 20th days of the treatment were: 58% and 168% compared with the intact group of animals and respectively, 313.5% and 601.2% compared with the control group. For (EPN), respectively 26% and 137%, and 230.5% and 522.6%. For (PSCh), respectively, 56.7% and 84.5%, and 310.4% and 383%.

Further, in the comparative aspect (Table 2) coefficient of variation from controls (CV) was determined for (F№1 + PhytoF), (EPN), (PSCh) by (CP), and the hepatoprotective activity was determined for the tested substances on the basis of data on the changes of the protein fractions.

As a result of the studies carried out to assess the direction of the changes of pharmacological effects, the tested compounds can be arranged in the following order according to biochemical parameters:

According to the influence on the synthesis of ceruloplasminthe studied substances act in the following order (CV):

-on the synthesis of CP from intact group on the 10th day (F№1 + PhytoF)> (EPN)> (PSCh)

-on the synthesis of CP from intact group on the 20th day (F№1 + PhytoF)> (EPN)> (PSCh)

-on the synthesis of CP from control group on the 10th day (F№1 + PhytoF)> (PSCh)> (EPN)

-on the synthesis of CP from control group on the 20th day: (F№1 + FPhyto)> (EPN)> (PSCh)

-on the total protein synthesis (EPN)> (PSCh)> (F№1 + PhytoF) on the 10th day;

 - on the total protein synthesis (EPN)> (F№1 + PhytoF)> (PSCh) on the 20th day;

- on the albumin synthesis: (PSCH)> (EPN)> (F№1 + PhytoF) on the 10th day;

- on the albumin synthesis (EPN)> (F№1 + PhytoF)> (PSCh) on the 20th day;

  - on the synthesis of globulins (F№1 + PhytoF)> (PSCh)> (EPN) on the 10th day;

- on the synthesis of globulins (F№1 + PhytoF)> (EPN)> (PSCh) on the 20th day;

- on the protein coefficient (PC) (F№1 + PhytoF)> (PSCh)> (EPN) on the 10th day;

  - on PC: (F№1 + PhytoF) = (EPN)> (PSCh) on the 20th day.

Hepatoprotective activity coefficient (HAC) was determined by the formula: HAC = [(Ppat-Pexp) / (Ppat- Pint)] (Table 1).

Ppat- arithmetic mean of the parameter in the group of animals with the toxic hepatitis;

Pexp- arithmetic mean of the parameterfor the animals in the experimental group;

Pint- arithmetic mean of the parameter of the group of intact animals.

HAC for (F№1 + PhytoF) according to the total protein synthesis was 0.39 on the 10th day of the treatment; 0.58 on the 20th day; albumins are respectively, 0.58 and 0.8; globulins 1.04 and 1.14.Protein coefficient was respectively 1.09 and 1.52. HAC for (EPN): total protein-0.57 and 0.7; albumins-0.61 and 0.9; globulins – 0.004 and 0.89; PК - 0.82 и 1.52, respectively.

HAC for (PSCh): total protein 0.5 and 0.375; albumins - 0.64 and 0.49; globulins– 0.64 and 0.55; PC -1.02 and 0.79. (Tab. №1)

Hepatoprotective activity coefficient (HAC) and protein coefficient (PC) according to protein metabolism before and after treatment, the total extract Phytocomposition №1 + Phyto F., Essenciale forte H and extract Paliurusspina - christi in toxic liver damage in rabbits

Table 1

Parameters Phyto composition №1 +Phyto F   Essenciale forte H     Paliurus spina - christi    
10 d   20 d   Average HAC 10 d   20 d   Average HAC 10 d   20 d   Average HAC
Total protein 0.39 0.58 0.48 0.57 0.7 0.64 0.5 0.375 0.44
Albumins 0.58 0.8 0.69 0.61 0.9 0.76 0.64 0.49 0.57
Globulins 1.04 1.14 1.09 0.004 0.89 0.45 0.64 0.55 0.596
Protein coefficient 1.09 1.52 1.3 0.82 1.52 1.17 1.02 0.79 0.9

As a result of the research, pharmacological compounds can be arranged in the following hepatoprotective activity order based on the changes of the protein fractions:

-on total protein synthesis (EPN)> (PSCh)> (F№1 + F) on the 10th day;

- on total protein synthesis (EPN)> (F№1 + F)> (PSCh) on the 20th day;

- on the albumin synthesis: (PSCH)> (EPN)> (F№1 + F) on the 10th day;

- on the albumin synthesis (EPN)> (F№1 + F)> (PSCh) on the 20th day;

- on the synthesis of globulins (F№1 + F)> (PSCh)> (EPN) on the 10th day;

-on the synthesis of globulins (F№1 + F)> (EPN)> (PSCh) on the 20th day;

- on PC: (F№1 + PhytoF)> (PSCh)> (EPN) on the 10thday;

 - on PC: (F№1 +PhytoF) = (EPN)> (PSCh) on the 20th day.

CV values and the statistical processing of the experimental study have been presented in tables 1, 2 :

Parameters of synthesis of ceruloplasmin (CP) and its correction by Phyto composition №1 + PhytoF, Essenciale Forte H and extract PaliurusSpinachristiin the experiment with modeling acute toxic liver injury in rabbits.

Table 2

Studied parameter Control (pathology) Temporary stages of the treatment
Prophylactics 10 days 20 days
CP /F№1+Phyto F 11.88  (+ -)  4.99**                                               (2.19 – 10.5) 24,99  (+ -)  2,81* (18.8 – 32.1) 26.83  (+ -)  5.24* (11.46 – 40.86) 44.72  (+ -)  5.89* (26.25 - 66)
CP / Spina christa 11.88  (+ -)  4.99** (2.19 – 10.5) 22.92  (+ -)  3.14* (14.4 - 32) 25.44  (+ -)  2.59* (18.6 – 32.2)
CP / Essenciale forte 12.16  (+ -)  5.28** (2.19 – 10.5) 26.44  (+ -)  2.52* (19.9 - 31) 20.99  (+ -)  3.5* (10.5 – 28.8) 39.61  (+ -)  3.97* (29.4 - 49)

NOTE: The first line contains the confidence intervals. (* With P <0.01; ** - p <0.05).

The second line - the minimum and maximum values of the parameter.

Dynamics of CV for the protein metabolism (F№1 + PhytoF) on the content of total protein after 10 days of treatment was 85.6% - from the intact group and the comparison group - 112%; after 20 days of treatment, respectively, 90.16% and 118%;

-on the synthesis of albumins, respectively: 83.6% and 136.7%; 92.4% and 151%;

- on the synthesis of globulins, respectively: 84.2% and 91.26%; 83.4% and 90.4%.

Dynamics of CV for the protein metabolism (EPN) on the content of total protein after 10 days of the treatment was 89.8% - from the intact group and the comparison group - 117.6%; after 20 days of treatment, respectively, 93.4% and 121.9%;

- on the synthesis of albumins, respectively, 85% and 139%; 96.2% and 157.3%;

- on the synthesis of globulins, respectively and 92.3% and 100%; 85.4% and 92.5%.

Dynamics of CV for the protein metabolism (PSCh) on the content of total protein after 10 days of the treatment was 88.3% - from the intact group and the comparison group - 115%; after 20 days of treatment, respectively, 85.2% and 111.6%;

- on the synthesis of albumins respectively 86.2% and 141%; 80% and 130%;

- on the synthesis of globulins respectively 87.3% and 94.6%; 88% and 95.4%.

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Thus, according to the results of research,hepatoprotective action confirms the correctness of the content of the total extract of Phyto composition №1 + PhytoF, which improves the synthesis of proteins and CP by hepatocytes. When comparing activity (F№1 + PhytoF) with the known (EFN), marked superiority on the side of (F№1 + PhytoF) was observed at (p <0.01). When comparing (F№1 + PhytoF)with the extraction of (PSCh), there was a significant (p <0.01) improvement in the function of protein synthesis and the CP synthesis in favor of “Phyto composition №1 + Phyto F”, but when comparing (PSCh) with (EFN ), then (PSCh) concedes to (EPN). Analyzing the results, we can assume the possible process of regeneration of hepatocytes under the influence of Phyto composition №1 + Phyto F.In addition, CP having radioprotective, immunomodulating, immuneprotector [9,11,15,12], antioxidant properties, increases the resistance of hepatocytes [5,6] to damaging toxic factors. Thanks to the ferroxidase activity, CP prevents the formation of free radicals and the development of lipid peroxidation (LPO) [11,13, 14]. In our studies, (F№1 + PhytoF) increased the CP synthesis on CV, according to the double control the extract has a leading position in comparison with the (EPN) and (PSCH). CP in this study has antioxidant and hepatoprotective effect against the toxic effects of carbon tetrachloride on hepatocytes.

Sum of flavonoids has an effective antioxidant (AO) activity from a number of natural cerealsin the composition ofF№1 + PhytoF.These areCalamagrostisepigejos, Daschampsiacaepitosa studied in our experiments [8]. Very small doses in terms of routines inhibit 30% of the chemiluminescence reaction (CL), and they posses not only highly AO, but also a high antiradical activity towards the radicals in the DPPH reaction [8]. Because of these properties, the condition of the animals improved in the experiment, their endurance to the toxic effects increased and survival increased.


As a result of studies it was found:

- Phytocomposition №1 + Phyto F is biologically active and it restores the synthetic function of hepatocyteson the production of CP;

- Hepatoprotective effect (F№1 + PhytoF) appears rapidly due to antioxidant and antiradical actions;

- 100% survival of the animals was established;

- Recovery of the total protein synthesis and globulin fraction occurred due to increasing synthesis of CP.


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