A Novel Molecular Approach for Enhancing the Safety of Ozone in Autohemotherapy and Insights into Heme Pocket Autoxidation of Hemoglobin.

Major ozone autohemotherapy (MAH) is a popular clinical practice for treating a variety of pathological conditions due to the mild and controlled oxidative stress produced by the reaction of ozone gas with other biological components. Previous studies have shown that blood ozonation leads to structural changes in hemoglobin (Hb); therefore, in the present study, the molecular effects of ozonation on Hb of a healthy individual were assessed by ozonating whole blood samples with single doses of ozone at 40, 60, and 80 µg/mL or double doses of ozone at 20 + 20, 30 + 30, and 40 + 40 µg/mL ozone to investigate whether ozonating once versus twice (but with the same final ozone concentration) would have varying effects on Hb. Additionally, our study aimed to verify whether using a very high ozone concentration (80 + 80 µg/mL), despite mixing it with blood in two steps, would result in Hb autoxidation. The pH, oxygen partial pressure, and saturation percentage of the whole blood samples were measured through a venous blood gas test, and the purified Hb samples were analyzed using several techniques including intrinsic fluorescence, circular dichroism and UV-vis absorption spectroscopies, SDS-polyacrylamide gel electrophoresis, dynamic light scattering, and a zeta potential analyzer. Structural and sequence analyses were also used to study the Hb heme pocket autoxidation sites and the residues involved. The results showed that the oligomerization and instability of Hb can be reduced if the ozone concentration to be used in MAH is divided into two doses. Indeed, our study demonstrated that two-step ozonation with 20, 30, and 40 µg/mL of ozone instead of single-dose ozonation with 40, 60, and 80 µg/mL of ozone reduced the potential adverse effects of ozone on Hb including protein instability and oligomerization. Moreover, it was found that for certain residues, their orientation or displacement leads to the entry of excess water molecules into the heme moiety, which can contribute to Hb autoxidation. Additionally, the autoxidation rate was found to be higher in alpha globins compared to beta globins.

Molecular effects of ozone on amino acids and proteins, especially human hemoglobin and albumin, and the need to personalize ozone concentration in major ozone autohemotherapy.

Major ozonated autohemotherapy is a complementary therapy that is widely used to treat various diseases. In the ozonation method, ozone that is dissolved in the plasma immediately reacts with biomolecules and produces H2O2 and lipid oxidation products (LOPs), which serve as ozone messengers/signaling molecules and result in the biological and therapeutic effects from ozonation. These signaling molecules affect hemoglobin and albumin, the most abundant proteins in red blood cells and plasma, respectively. Because hemoglobin and albumin perform important physiological functions, structural changes due to complementary therapeutic procedures and interventions such as major ozonated autohemotherapy at incorrect concentrations can lead to disruption of their functions. Oxidation reactions in hemoglobin and albumin can lead to unfavorable high molecular weight species, which can be prevented through personalized and correct use of ozone concentrations. In this review, we describe the molecular aspects of the effects of ozone on hemoglobin and albumin at inappropriate concentrations, which cause oxidation reactions that result in destructive effects; discuss the potential risks when ozonated blood is re-infused into the patient's blood stream in the process of major ozonated autohemotherapy; and emphasize the need for personalization of ozone concentrations.

Highlighting the Importance of Water Alkalinity Using Phosphate Buffer Diluted With Deionized, Double Distilled and Tap Water, in Lowering Oxidation Effects on Human Hemoglobin Ozonated at High Ozone Concentrations in vitro.

In autohemotherapy, it is important to find a way to lower the effects of oxidation, especially at high concentrations of ozone. One of the parameters, other than ozone concentration, which can have a significant effect on the stability and rate of decomposition of ozone at high concentrations, is the presence of ions in water. A number of spectroscopic techniques including intrinsic fluorescence, circular dichroism and UV-VIS were used as well as SDS-PAGE, Native-PAGE dynamic light scattering and water ion analysis, in order to investigate the effects of two relatively high concentrations of ozone on purified human hemoglobin (Hb) in phosphate buffer and diluted versions with deionized, double distilled and tap water in vitro. Purified human Hb and not whole blood human Hb was used in this study, since the addition of water to the whole blood would have caused the RBCs to lyse, affecting the purification of Hb for further analysis. Therefore, using purified Hb, it was possible to investigate the effects of dilution of 50 mM phosphate buffer to 10 mM phosphate buffer with different water types including non-ion containing deionized and double distilled water as well as ion-containing tap water, when ozonated at 55 and 80 µg/ml ozone. The fundamental changes in the secondary and tertiary structures of Hb were seen to be related to non-ozonated Hb samples diluted with deionized and double distilled waters, respectively. Generally, Hb oligomerization was more likely to occur at the higher concentration of ozone (80 µg/ml) and in samples where phosphate buffer was diluted with non-ion containing deionized and double distilled waters and not the ion-containing tap water. This could be explained by the presence of water alkalinity or bicarbonate ions in tap water, which can scavenge free radicals and reduce Hb oxidation/oligomerization. Therefore, it was concluded that Hb could best withstand high concentrations of ozone in the presence of the undiluted 50 mM phosphate buffer followed by phosphate buffer diluted with tap water, containing bicarbonate ions.

Anticancer activity of Astragalus ovinus against 7, 12 dimethyl benz (a) anthracene (DMBA)-induced breast cancer in rats.

OBJECTIVE: Some species of Astragalus are used for the treatment of various types of cancer. The present study was designed to evaluate the anticancer potential of Astragalus ovinus extract (AOE) against DMBA-induced breast carcinoma in rats. MATERIALS AND METHODS: The anti-tumor and antioxidant effects of AOE were evaluated against DMBA-induced breast carcinoma in rats using DPPH, FRAP and ABTS technique, respectively. Forty adult female Sprague-Dawley rats were randomly divided into four groups including the control group received a single dose of DMBA solvent orally, and groups II, III and IV received a single dose of DMBA (40 mg/kg) dissolved in olive oil. Groups I and II received normal saline and groups III and IV were treated with AOE orally (120 and 240 mg/kg respectively) for 60 consecutive days. Chemopreventive effects were assessed in terms of diameter and volume of tumors, expression levels of PCNA, and serum levels of CA15.3, p53, MDA, CAT, and calcium, and histopathological features. RESULTS: AOE contained a noticeable amount of phenolic and flavonoids compounds. This extract showed a potent antioxidant activity both in vitro and in vivo. AOE significantly decreased the diameter and volume of tumors (p<0.01) and reduced the serum levels of CA15.3 (p<0.001), p53 (p<0.01), MDA (p<0.001), and calcium (p<0.01). AOE also decreased the expression of PCNA in cancerous tissues and reduced the histopathological deformity. CONCLUSION: According to the data, AOE produced a significant chemopreventive activity in DMBA-induced breast tumors in rats, probably due to its antioxidant and its inhibitory effect on some tumorigenicity markers such as CA15.3, p53 and PCNA activity.

Personalizing the safe, appropriate and effective concentration(s) of ozone for a non-diabetic individual and four type II diabetic patients in autohemotherapy through blood hemoglobin analysis.

BACKGROUND: Diabetes is a chronic disease associated with many problems and high costs. In recent decades, a lot of research has been carried out in order to improve methods of treatment of diabetic patients. One of the currently used complementary therapies for diabetes is ozone therapy or autohemotherapy. The beneficial effects of ozone has been proven in many diseases such as diabetes, but the critical issue is the determination of the safe and effective concentration of ozone reacting with blood and in particular hemoglobin. METHODS: A number of spectroscopic techniques including intrinsic fluorescence, circular dichroism and UV-VIS spectroscopies were used as well as SDS-PAGE, Native-PAGE and dynamic light scattering to analyze the effect of ozonation on hemoglobin of a non-diabetic individual and four diabetic patients in order to find the appropriate concentration(s) of ozone for personalized autohemotherapy. RESULTS: In this study, we determined the personalized concentration(s) for a safe and effective ozonation of a non-diabetic individual and four diabetic type II patients, based on blood hemoglobin analysis. CONCLUSIONS: A number of techniques were used to determine the personalized ozone concentration(s) for a safe and effective autohemotherapy based on blood hemoglobin analysis. SDS-PAGE and dynamic light scattering were identified as the two main techniques needed for personalizing the ozone concentration(s) for each individual as otherwise hemoglobin in blood can oligomerise and cause serious damage if the inappropriate ozone concentration is used.

Novel X-ray sequences and crystal structures of Persian and Starry sturgeon methemoglobins: Highlighting the role of heme pocket waters in causing autoxidation.

Although there is a high sequence similarity between mammalian and fish hemoglobin (Hb), the oxidation and heme loss rates can vary greatly between them such that fish Hbs oxidise much more rapidly than mammalian Hbs. There is to date no sequence or structural data for any sturgeon Hb to reveal the level of autoxidation in these fish. In this study, novel high resolution X-ray sequences and crystal structures of methemoglobin (Met-Hb) from two sturgeon fish including Persian sturgeon (Acipenser percisus) and Starry sturgeon (Acipenser stellatus) belonging to the Caspian sea has been determined. A comprehensive sequence and structure comparison between these sturgeon Met-Hbs and a number of non-sturgeon and normal and sickle cell anaemia human Hb in varying heme states has been carried out highlighting (i) the structural variability in the heme propionate groups; (ii) the existence of certain residues or their displacement and shift in the heme pocket allowing entry of water molecules into the heme pocket; (iii) the importance of the number of water molecules in the heme pocket; (iv) the hydrogen bonding between oxygens of A and D propionate groups and that of waters in the heme pocket; and (v) the role of heme binding waters causing oxidative stress and heme autoxidation.

Molecular insights into the effect of ozone on human hemoglobin in autohemotherapy: Highlighting the importance of the presence of blood antioxidants during ozonation.

Ozone has been known for several decades, with its antiseptic and therapeutic effects determined by the hormesis theory. It is shown that the therapeutic efficacy of ozone therapy may be partly due to the controlled and moderate oxidative stress produced by the reaction of ozone with several biological components. In this study, the effect of ozone on healthy human hemoglobin (Hb) in the whole blood environment (in the presence of antioxidants) and in the purified form (in the absence of antioxidants) is investigated using a number of different techniques including intrinsic fluorescence, circular dichroism and UV-VIS absorption spectroscopy as well as SDS- and Native-PAGE and dynamic light scattering. The results show that the presence of antioxidants prevents damage to Hb while its absence means that as the exposure to ozone is increased, Hb is increasingly damaged. These results highlight the importance for the use of appropriate doses of ozone, for patients with different diseases and hence antioxidant levels, in autohemotherapy.

Effects of date palm pollen (Phoenix dactylifera L.) and Astragalus ovinus on sperm parameters and sex hormones in adult male rats.

BACKGROUND: Date Palm Pollen (DPP) and Astragalus genus are used in some countries for the treatment of infertility. OBJECTIVE: This study was designed to investigate effects of DPP and Astragalus ovinus (A.Ovinus) on fertility in healthy adult male rats. MATERIALS AND METHODS: Thirty-six rats were divided into six groups (n=6) including control and five treatment groups. DPP (120, 240 and 360 mg/kg) and A.ovinus (100, 500 mg/ kg) were orally given to the treatment groups. After thirty-five days, blood samples were taken to determine serum levels of FSH, LH, testosterone and estradiol. Weight of testis and epididymis, sperm count, sperm motility, seminiferous tubules diameter (STD), germinal cell layer thickness (GCLT), sertoli, leydig and spermatogonia cells were also evaluated. RESULTS: DPP at the of 120 and 240 mg/kg doses significantly raised the ratio of testis or epididymis to body weight, sperm count, sperm motility , and estradiol level compared to the control group (p<0.05). LH and testosterone levels only noticeably increased at 120 mg/kg of DPP (p<0.01 and p<0.001 respectively). STD increased in the three applied doses (p=0.001). A. ovinus extract at the indicated doses produced a significant reduction in the ratio of testis or epididymis to body weight and sperm motility (p<0.05). Sperm count, spermatogonia, leydig cells and FSH level decreased at dose of 500 mg/kg. Furthermore, GCLT, spermatogonia cells, and serum estradiol level increased at 100 mg/kg dose of A. ovinus. CONCLUSION: Our findings indicate that DPP could improve fertility factors, while A.ovinus can exhibit deleterious effects on gonad and sperm parameters in rats.

In vivo anti-inflammatory properties of aerial parts of Nasturtium officinale.

CONTEXT: Nasturtium officinale R. Br. (watercress) has long been used in Iranian folk medicine to treat hypertension, hyperglycemia, and renal colic. Moreover, anticancer, antioxidant, and hepatoprotective properties of N. officinale have been reported. OBJECTIVE: In this study, anti-inflammatory activity of the hydro-alcoholic extract from aerial parts of N. officinale was investigated. MATERIALS AND METHODS: Oral administration of the hydro-alcoholic extract of N. officinale (250, 500 and 750 mg kg(-1)) was investigated on two well-characterized animal models of inflammation, including carrageenan- or formalin-induced paw edema in rats. Then, the topical anti-inflammatory effect of N. officinale (2 and 5 mg/ear) was studied on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema. Finally, biopsy of the paw or ear was performed for pathological evaluation. RESULTS: Acute toxicity tests of N. officinale in rats established an oral LD50 of >5 g kg(-1). The extract of watercress (250, 500 and 750 mg kg(-1)) significantly inhibited carrageenan-induced paw edema 1, 2, 3 and 4 h after carrageenan challenge (p < 0.001). The extract (500 mg kg(-1)) also showed considerable activity against formalin-evoked paw edema over a period of 24 h (p < 0.001). Furthermore, topical application of N. officinale (5 mg/ear) reduced TPA-induced ear edema (p < 0.05). Histopathologically, the extract decreased swelling and the tissue damage induced by carrageenan or TPA. DISCUSSION AND CONCLUSION: Our findings indicate potent anti-inflammatory activity of N. officinale in systemic and topical application and propose its potential as an anti-inflammatory agent for treatment of inflammatory conditions.