Journal of Ethnopharmacology 90 (2004) 105114

Amrita Binduan antioxidant inducer therapy in asthma children

Sumathy S. Kumar, K. Radha Shanmugasundaram
Department of Medical Biochemistry, Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India Received 20 February 2003; accepted 22 September 2003

Abstract Studies all over the world on the therapeutic use of antioxidants as supplements has revealed their capacity to control inammatory processes. Amrita Bindu an Ayurvedic health food supplement has already shown to be an antioxidant inducer and to combat free radical-mediated tissue damage studied in rats. Amrita Bindu is a saltspice herbal mixture designed for positive health. It was tested as a supplement to therapy for a period of 12 months in 36 children suffering from asthma. Asthma is a chronic inammatory disease with excessive free radical generation in lungs and blood cells. The patients were followed up by monitoring their clinical conditions, therapeutic doses of anti-asthmatic drugs, free radical generation, lipid peroxidation (LPO) and antioxidants in blood. At the end of 3 months of Amrita Bindu supplementation, the patients had stopped all anti-asthmatic medications and were free from attacks of asthma. 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antioxidants; Asthma; Allergy; Free radicals; Inammation; Lipid peroxidation

1. Introduction Reactive oxygen species (ROS) can severely injure the lung. Lung is exposed to high concentrations of oxygen and its metabolites because of its large surface area (approximately 100 m2 in a 70 kg adult). ROS inhibit neutral endopeptidase activity in airway epithelium and aids in exaggeration of the bronchoconstrictor response to these peptide. In patients with asthma, oxidants present in the polluted air, exacerbate existing airway inammation (Barnes, 1990). Evidence of excessive free radicals formation (superoxide and hydroxyl) and elevated levels of lipid peroxidation (LPO) has been observed in the red blood cells of asthma children (Shanmugasundaram et al., 2001). These free radicals are detoxied by primary intracellular antioxidant enzyme systems comprising of superoxide dismutase (SOD), catalase activity (CAT), and glutathione peroxidase (GPx) (Sies and Stahl, 1995). Cross et al. (1994) have shown that the respiratory tract is lined with main antioxidants such as reduced glutathione (GSH), ascorbic acid, mucin, uric acid, metal binding proteins, antioxidant enzymes and sacricial proteins. Smith et al. (1997), reported reduced SOD activity in the lung cells of asthma patients. Antioxidant deciency
author. Tel.: +91-44-24480767; fax: +91-44-24926709. E-mail address: sunrays@md5.vsnl.net.in (K.R. Shanmugasundaram).
Corresponding

has been observed in the red blood cells of asthma children (Shanmugasundaram et al., 2001) which suggest that diminished antioxidant status served as a marker of inammation characterising asthma. Alternatively, it may play a role in the development or severity of the disease. Many studies all over the world showed that antioxidants served as thrapeutic agents in controlling inammatory responses (Romieu et al., 1998; Rust et al., 1998). One such antioxidant inducer therapy in our laboratory, aimed at augmenting natural antioxidants is Amrita Bindu. It is a saltspice herbal mixture, formulated based on the principles of Ayurveda and siddha medicine. Indian medicine advocates the intake of Jivanya (life sustaining) group of herbs and salts as food additives. According to the Caraka and Samhita (1st century a.d.) Jivanya includes invigorators, nourishing drugs, laxatives, wound healers and digestive stimulants. The second group is Balya or promoters of strength, complexion, voice and cardiac resilience to stress. The third are Sowdhanyas, meaning cleansers of the organ systems. They used to prevent haemorrhoids, skin diseases, inammation and infections. The fourth group of drugs clear the passages in the mammary gland and the seminal tract and the fth are used to promote secretions from eyes, nose and also for enemas. The sixth group includes antiemetics, and herbs that control thirst, while the seventh include bowel binders, antidiuretics and diuretics. The eighth group of drugs are curatives of dyspnoeas, oedema, fever and fatigue, the ninth

0378-8741/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2003.09.031

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is a set of pain relievers and the tenth group includes haematinics, sedatives and rejuvenators. In addition to common salt, Indian medicine advocates a variety of salts like rock salt, saindhawa and bamboo salt that provides chlorides, phosphates and sulphates of potassium and magnesium and black salt with sulphides and trace elements. Amrita Bindu has been formulated based on the above mentioned principles. It was found to combat free radical insults and to provide protection against LPO and antioxidant depletion in nitrosamine-induced tissue damage studied in albino rats (Shanmugasundaram et al., 1994). Amrita Bindu has also been tested as a supplement in diabetic retinopathy and has been found to raise the antioxidant levels in blood and prevent further damage of the retina (Shanmugasundaram et al., 1996). The present study was undertaken to assess the effects of Amrita Bindu supplementation in combating free radicals and to act as an antioxidant inducer during asthma. The efciency of the Amrita Bindu in effectively scavenging free radicals and its antioxidant potential were studied under in vitro conditions. 2. Methodology 2.1. Subjects investigated The studies were conducted on blood samples obtained from asthma children in the age group of 518 years, who were patients at Childs Trust Hospital, Nungambakkam, Chennai 600 034. They were mainly from middle income (socio-economic) groups and were under the therapeutic care of Dr. Sarala Rajajee and Dr. Janani (Chief Paediatricians). Assessment of lung function tests, therapeutic measures, clinical examinations were carried out by the medical and paramedical staff in the centre. 2.2. Criteria for diagnosis of asthma The children presented with wheezing, dyspnoea or difcult breathing. They had experienced asthma episodes many times in the past, and were on medication. Every one of them had a history of repeated respiratory infections, with wheezing heard when auscultating the chest. In some chronic sufferers with dyspnoea/difcult breathing, wheezing was audible after forced expiratory manoeuvre during pulmonary function testing, with a prolonged expiratory phase suggesting obstruction in the airways. Evidence of upper airway sounds, viscous and more tenacious sputum were the other symptoms seen in this group to conrm asthma (Farr, 1985). Thirty-six patients having severe episodes of wheezing, who often had regular attacks of asthma, with poor response to usual bronchodilators and systemic steroids were involved in our clinical trial to assess the therapeutic value of a saltspice herbal preparation AMRITA BINDU as a health food supplement for a period of 12 months. Twenty-two chil-

dren (12 males,10 females) in the age group of 510 years were administered 250 mg Amrita Bindu twice daily mixed with 5 ml honey after food. Fourteen children (8 males, 6 females) of 1118 years were administered 500 mg Amrita Bindu in hard gelatin capsules, twice daily after food. They continued with all other medications. Informed consents were obtained from the children and their parents and the study was approved by the Institutional Ethical Committee. Amrita Bindu is prepared by Prof. E.R.B. Shanmugasundaram, Herbal Research Foundation, Chennai. Amrita Bindu is a saltspice herbal mixture and the ingredients present in it are (1) Induppoo or saindhawa, (2) Kalluppoo (rock salt), (3) Moongiluppoo (pearl ash), (4) Valayaluppoo (bangle salt), (5) Karuuppoo (black salt), (6) Vengaram (crude borax), (7) Tribulus terrestris L. (small caltrops), (8) Calatropis gigantean R.Br. (gigantic swallow wort and mudar), (9) Zingiber ofcinale (ginger), (10) Piper longum L. (dried catkins and long pepper), (11) Piper nigrum L. (black pepper), (12) Plumbago zeylanica L. (Ceylon leadwort), (13) Cyperus rotundus L. (nutgrass). Its formula and its preparation is described in detail by Shanmugasundaram et al. (1994). Amrita Bindu is reported to have no hepato or nephrotoxicity when administered for long periods. Thirty patients (18 in the age group of 510 years and 12 in the age group of 1118 years) were followed up for a period of 12 months. Blood analyses were made at the end of 1, 3, 6, 9 and 12 months of Amrita Bindu supplementation. Four patients could not be followed up due to poor adherence to medication and two did not come back to the hospital. There were six drop outs in the Amrita Bindu supplemented group. Fifty-two healthy children (32 children, 18 males, 14 females in the age group of 510 years; and 20 adolescents, 12 males, 8 females in the age group of 1118 years) from Government school, Nandanam, Chennai 600 035 were also included in the study as age and sex matched healthy controls (H.C.). None of the children smoked cigarettes or used any form of tobacco. A standardised Child Health Questionnaire (CHQ) was used to obtain all the information on respiratory illness of the children and their parents, frequency of spacers/inhalers used, drugs prescribed to children and on family background. Anthropometric measurements of all the children were taken. The lung function tests (peak expiratory ow rate) were performed at the Childs Trust Hospital, Chennai, using Wright Peak Flowmeter. Each child blew into the Wright Peak Flowmeter three times and the highest reading was accepted in each case. Repeat values were obtained from among one third of the volunteers on two different occasions, within 10 days. The variation was less than 10%. All the subjects in this group continued with their usual diets and other activities. Medications were reduced/discontinued depending on the clinical conditions at the time of periodical monitoring. Records were maintained on the dosage and frequency of the usage of anti-asthmatic drugs. These children were examined by doctors who classied as severe asthmatics in accordance with criteria for diagnosis of asthma.

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107

2.3. Blood analyses Fasting blood samples were drawn by venipuncture and aliquots were transferred to (a) tube containing EDTA (1 mg/ml) for plasma and cell separation, (b) a tube containing 1 mM diethyl dithiocarbamate (DDC) to inhibit the activity of SOD and measure superoxide formation. The blood samples were packed in ice during transportation to the laboratory and analysis was initiated immediately. 2.4. Assays made in whole blood and plasma Haemoglobin was assayed in whole blood by its oxidation to methaemoglobin with alkaline ferricyanide reagent giving intensely coloured cyanmethaemoglobin. Blood glucose was estimated by the O-toludine method (Dubowski, 1962). GSH (Beutler and Kelly, 1963) and Vitamin C (Omaye et al., 1979) were assayed in whole blood. Plasma was separated (from blood samples collected with EDTA as anti-coagulant) by centrifugation at 4000 rpm for 10 min. Vitamin A (Bayeld and Cole, 1980) and Vitamin E (Taylor et al., 1976) were assayed in plasma. 2.5. Antioxidant assays and lipid peroxidation measurement The red cells were washed thrice with isotonic saline to remove the buffy coat and then sedimented. Cells were lysed with ice cold water and membrane sedimented according to the method of Dodge et al. with modications reported earlier by Shanmugasundaram et al. (1992). SOD (Marklund and Marklund, 1974) and GPx (Rotruck et al., 1973) were assayed in the haemolysate. CAT (Sinha, 1972) and membrane protein content (Lowry et al., 1951) were determined in the cell membrane. The product of LPO was measured as TBARS and expressed in terms of malondialdehyde (MDA) in the plasma (Yagi, 1976), haemolysate and erythrocyte membrane (Cynamon et al., 1985). 2.6. Measurements of superoxide and hydroxyl free radicals Erythrocytes were lysed immediately, by mixing together 0.5 ml of cells and 10 mM sodium phosphate buffer, pH 7.4 containing 67 mM NaCl, and 0.1 mM EDTA. Thus, the haemolysate prepared were divided into two aliquots to measure superoxide and hydroxyl radicals. The superoxide radical was determined in terms of the reduction of nitroblue tetrazolium (NBT) to formazan according to the method of Nishikimi et al. (1972). To one aliquot added 0.2 ml of 4 mM NBT containing 340 mM sucrose and 2.0 ml of 0.1 M sodium hydroxide in 24 mM sodium hydrogen carbonate. The blue insoluble formazan was dissolved in 5.0 ml of 1,4-dioxan and read spectrophotometricaly. For each subject, one sample of cells collected with SOD inhibited DDC, and one sample of cells without DDC were processed simultaneously

and NBT reduction was measured. The difference between the two is a measure of superoxide radical present at that time. The hydroxyl radical in the haemolysate was measured by the method of Gutteridge (1981) in which the hydroxyl radical reacts with 2-deoxyribose. The resulting complex mixture of products was heated under acid conditions, and the degradation products were measured as thiobarbituric acid reactive species (TBARS). 2.0 ml of 20 mM 2-deoxyribose was added to the other aliquot and the mixture was incubated at 37 C for 1 h. 0.5 ml of thiobarbituric acid (TBA) and 0.5 ml of trichloroacetic acid (TCA) were added and heated in a boiling water bath for 1015 min, cooled and read spectrophotometrically. Controls were included in which 2-deoxyribose was omitted, and the residual thiobarbituric reactive acid species was measured as haemolysate. The difference between the two readings gave the hydroxyl content at that time. 2.7. Assessment of free radical scavenging property of Amrita Bindu Amrita Bindu was tested for its ability to neutralise superoxide and hydroxyl radicals formed in Fentons reagent. Fentons reagent was an aqueous solution of hydrogen peroxide (110 mM) containing Fe2+ at 30 M. Fentons reaction: Fe3+ + O2 Fe2+ + O2 Fe2+ + H2 O2 Fe3+ + OH + OH

2.8. Superoxide and hydroxyl scavenging action by Amrita Bindu Superoxide and hydroxyl ions were generated in Fentons mixture (Fe2+ + H2 O2 ). Ten millilitres of incubation mixture were prepared by adding 1 ml of 300 M FeSO4 and 1 ml of hydrogen peroxide (10100 mM) to 8 ml of 0.01 M phosphate buffer at pH 7.4. In a duplicate set of tubes, 100 mg of Amrita Bindu suspended in the buffer was taken instead of buffer. The mixture was incubated at 37 C for different time intervals (15, 30, 45 and 60 min). To 4 ml of aliquot, 0.2 ml of NBT and 2.0 ml of sodium hydroxide were added. The contents were centrifuged and the precipitate was dissolved in 5 ml of 1,4-dioxan. The optical density was read at 520 nm. Each determination was duplicated. The superoxide radical present in the incubation mixture and superoxide radical scavenging capacity of Amrita Bindu were determined. In several tubes taken in duplicate 1ml of 300 M FeSO4 solution was mixed with 1 ml of 0.01 M sodium phosphate buffer containing 20 mM deoxyribose and 8 ml of hydrogen peroxide (10100 mM) in order to constitute 10 ml of the reaction mixture. In one set of tubes, the buffer contained 100 mg of Amrita Bindu in suspension. The mixture was incubated at 37 C for different time intervals (15, 30,

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45 and 60 min) and 1% TBA followed by 2.8% TCA were added. The contents were heated in a boiling water bath for 1015 min, cooled and optical density was read at 532 nm. Hydroxyl radical generation and hydroxyl radical scavenging capacity of Amrita Bindu were determined. 2.9. Assessment of the combined antioxidant and free radical scavenging properties of Amrita Bindu This study was designed to nd out the free radical quenching and combating capacity of Amrita Bindu by incubation with plasma and measuring the LPO products for the next 60 min. Blood samples were obtained from 10 asthma and 10 healthy children. The pooled plasma samples were taken as substrate. To 2.0 ml of pooled plasma (asthma and healthy children separately), was added 2 ml of supernatant obtained by dissolving 100 mg of Amrita Bindu in phosphate buffer and centrifuging. The mixture was incubated at 37 C for different time intervals (0, 15, 30, 45 and 60 min). Phosphate buffer without Amrita Bindu was taken as a blank. The LPO levels were measured as TBARS. 2.10. Expression of cell and membrane constituents per 1012 cells The cell membrane constituents (catalase, LPO products) and free radical production (superoxide and hydroxyl radicals) are expressed per 1012 cells. This was arrived at from the cell count, haemoglobin in the lysate and membrane protein measurements. From the total count and haemoglobin in lysate, the number of packed cells per millilitre were calculated. 2.11. Statistical analysis The values are expressed as mean S.D. for the asthmatic patients and healthy population separately. Statistically signicant differences was arrived at using students two-tailed t test.

3. Results Table 1 shows the general data of 30 children who participated in the clinical trial of Amrita Bindu supplementation for 12 months. These children were the regular out patients of Childs Trust Hospital and were graded severe asthmatics by the chief pediatrician. Twenty-two children were constantly using salbutamol nebuliser/inhaler (200 g per puff) atleast three times a day. Twelve children were taking beclomethasone, daily doses of 400650 g. In addition, 14 children had asthalin, 2 mg twice daily. Eight children in the age group of 1118 years were taking theophyline, 10 mg a day. These children were coughing, wheezing and had respiratory distress over a period of 35 days a week. They never responded to cough expectorants and showed poor compliance to these anti-asthmatic drugs. They were regularly missing their school days and attending the hospital for medical treatment. After a month, children responded well to the supplementation and started reducing their frequency of dosage of anti-asthmatic drugs to once a day. Three months after the supplementation of Amrita Bindu, cough and colds subsided, frequency of wheezing was reduced to occasional and these occasional episodes were very mild. Twenty-ve children out of 30 became symptom free after 3 months and discontinued their usage of salbutamol nebulisers/spacers and bronchodilators. Their lung capacity also improved (measured by Wright Peak Flowmeter). The remaining ve had reduced their salbutamol inhalation to once a day (50 g per puff). All ve had discarded salbutamol at the end of 9 months supplementation. Table 2 presents the effects of Amrita Bindu supplementation on general biochemical parameters in 30 asthmatic children (18 in 510 years and 12 in 1118 years age group) parameters that were assayed to check for any adverse effects. Blood haemoglobin levels were found to increase gradually by about 13% over a 12-month period of supplementation. Amrita Bindu supplementation did not produce any change in the blood glucose or non-protein nitrogen, indicating that it has no toxicity to the islets of langerhans and kidney.

Table 1 General clinical and physical data on the population investigated (mean S.D.) Parameters 510 years Healthy controls (n = 18) Age (years) Height (cm) Weight (kg) BMI (kg/m2 ) Duration of asthma (years) Males (%) Percentage of patients using spacers/inhalers Family historya of asthma (%) Family history of smoking (%)
a

1118 years Asthmatics (n = 18) 72 118 9 18 7 12.9 2 25 61 80 55 70 Healthy controls (n = 12) 15 4 152 8 43 8 18.6 2.1 45 14 21 Asthmatics (n = 12) 15 3 144 7 37 6 17.8 2.3 712 33 62 58 64

73 124 7 22 9 14.3 2.4 63 9 30

Atleast one parent having the disease.

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105114 Table 2 Effect of Amrita Bindu supplementation on biochemical parameters in severe asthmatics (mean S.D.) Whole blood Hb (g/l) 510 years (n = 18) Initial 1 month 3 months 6 months 9 months 12 months Healthy controls 1118 years (n = 12) Initial 1 month 3 months 6 months 9 months 12 months Healthy controls

109

Serum Glu (mmol/l) 4.9 4.8 4.8 4.4 4.5 4.2 0.2 0.3 0.4 0.2 0.4 0.3 HbA1 C (%) 3.7 3.9 4.1 3.7 3.6 3.4 0.3 0.2 0.3 0.4 0.2 0.3 Urea (mmol/l) 3.4 3.5 3.3 3.1 3.1 3.1 0.1 0.2 0.3 0.3 0.4 0.3 Uric acid (mol/l) 122 126 131 128 133 135 7 5 6 9 8 9 Creatinine (mol/l) 36 37 37 35 36 35 4 5 6 7 4 3

125 127 131 137 139 141

4 3 4 3 5 2

136 9 130 132 136 140 148 151 6 5 7 9 6 4

4.1 0.4 5.6 5.5 4.9 4.4 4.6 4.3 0.4 0.5 0.6 0.7 0.5 0.4

3.9 0.7 4.7 4.6 4.4 4.5 4.4 4.3 0.2 0.3 0.2 0.4 0.4 0.2

3.0 0.2 3.7 3.6 3.5 3.4 3.4 3.2 0.3 0.3 0.4 0.2 0.4 0.3

127 18 146 148 152 157 163 172 14 12 15 16 11 13

38 7 39 38 37 39 38 39 4 5 6 7 4 5

145 11

4.7 0.6

4.3 0.6

3.3 0.3

167 30

43 9

Statistically signicant differences from the initial level at the lowest month of supplementation: P < 0.05.

Amrita Bindu supplementation raised the lung capacity as measured by peak expiratory ow rate to about 65% almost reaching the levels of controls (Table 3 measured at the intervals of 6 months). Table 3 gives the level of superoxide and hydroxyl radicals formation in the red blood cells in children during supplementation. Signicant decreases in the level of both free radicals were noticed after 3 months of supplementation, while superoxide radical generation reached a normal level (compared to healthy subjects) in 12 months, hydroxyl radiTable 3 Effect of Amrita Bindu supplementation on free radical formation in red blood cells, and PEFR of asthma children (mean S.D.) Superoxide (mmol NBT/1012 cells) 510 years (n = 18) Initial 108 1 month 97 3 months 74 6 months 58 9 months 44 12 months 32 Healthy controls 7 4 5 3 4 6 Hydroxyl (nmol MDA/1012 cells) PEFR (l/min)

cal generation reached normal levels at the end of 9 months therapy. Table 4 shows the LPO levels in the blood of asthmatic children treated with Amrita Bindu for a period of 12 months. It was observed that supplementation of Amrita Bindu led to a steady decrease in the levels of LPO product in plasma, RBC lysate and RBC membranes. LPO products in plasma had decreased by 48% and had reached the control values after 9 months of Amrita Bindu supplementation. Reduction in the TBARS in the haemolysate as well as in the membrane fraction were much greater after Amrita
Table 4 Effect of Amrita Bindu supplementation on LPO products (TBARS) expressed as MDA in blood of asthma children (mean S.D.) Plasma (nmol/dl) 510 years (n = 18) Initial 328 1 month 312 3 months 276 6 months 232 9 months 197 12 months 172 Healthy controls 15 20 22 19 21 15 Cell lysate (nmol/1012 cells) 26 24 21 16 13 10 2 2.5 1.5 2.0 1.8 1.5 Membrane (nmol/1012 cells) 546 522 487 402 342 285 84 68 75 53 62 46

14 12 8 5 3 3

2 3 2 2 1.5 1

108 21 136 30 178 36 235 37 201 18 247 33 286 26 263 47

38 8

3 1.5 27 24 18 11 8 4 3 2 3 4 3 2.5

163 16

9 2.0 29.6 3 27 3.5 25 3.5 21 2.2 18 2 13 1.5 10.5 1.5

235 37 656 642 587 498 405 325 96 55 62 71 57 66

1118 years (n = 12) Initial 108 7 1 month 97 4 3 months 74 5 6 months 58 3 9 months 44 4 12 months 32 6 Healthy controls

1118 years (n = 12) Initial 342 21 1 month 320 20 3 months 280 28 6 months 235 22 9 months 200 25 12 months 174 18 Healthy controls

38 8

4.6 2

171 20

242 28

Statistically signicant differences from the initial level at the lowest month of supplementation: P < 0.05.

Statistically signicant differences from the initial level at the lowest month of supplementation: P < 0.05.

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Table 5 Effect of Amrita Bindu supplementation on antioxidant enzymes in blood of asthma children (mean S.D.) SOD (IU/mg Hb) 510 years (n = 18) Initial 1.94 1 month 2.11 3 months 2.80 6 months 3.42 9 months 3.55 12 months 3.61 Healthy controls GPx (g GSH/mg Hb/min) 5.1 5.4 5.7 6.0 6.3 6.5 0.3 0.4 0.2 0.3 0.3 0.4 CAT (mmol H2 O2 /1012 cells/min) 3.9 4.1 4.5 4.6 4.8 4.9 0.20 0.22 0.3 0.25 0.30 0.28

to rise in the Vitamins A, C, E and GSH levels compared to those seen in healthy age matched children. 3.1. Assessment of free radical scavenging and total antioxidant property of Amrita Bindu Amrita Bindu was tested for its ability to scavenge superoxide and hydroxyl radicals formed in the Fenton reaction. Amrita Bindu added to the reaction mixture was able to lower the formation of superoxide radical. The percentage inhibition of formation of superoxide radical was more than 63%, and the effect was more pronounced at 15 min when whole of Amrita Bindu was used (Fig. 1). Similar observations were seen with hydroxyl formation in Fenton reaction with and without Amrita Bindu (Fig. 2). These observations indicate that Amrita Bindu can effectively scavenge and intercept the free radicals, superoxide and hydroxyl radicals formed in the reaction mixture. The overall antioxidant property of Amrita Bindu was also assessed by measuring the LPO products over 60 min (Fig. 3). Pooled plasma obtained from 10 healthy children and 10 asthmatic children, served as the substrate. LPO measured as TBARS showed a marginal decrease in healthy samples, whereas decreases in the order of 40 nmol/dl were noted in asthma samples after 60 min with Amrita Bindu. In the case of blanks without Amrita Bindu, LPO levels increased after 1 h. These results show that Amrita Bindu act as a free radical quencher and can combat the free radicals as and when they are formed in this system.

0.26 0.32 0.23 0.21 0.15 0.20

3.64 0.13

6.66 0.3 4.6 4.8 5.2 5.8 6.1 6.4 0.4 0.3 0.2 0.3 0.3 0.2

4.85 0.31 3.3 3.4 3.9 4.2 4.4 4.5 0.31 0.42 0.21 0.33 0.15 0.23

1118 years (n = 12) Initial 1.71 0.24 1 month 1.92 0.22 3 months 2.42 0.3 6 months 2.91 0.25 9 months 3.30 0.30 12 months 3.46 0.22 Healthy controls

3.55 0.13

6.3 0.3

4.51 0.26

Statistically signicant differences from the initial level at the lowest month of supplementation: P < 0.05.

Bindu supplementation, they were reduced almost to half the initial high value at the end of 1-year period of Amrita Bindu supplementation. Statistically signicant decrease in the levels of LPO product were observed after 3 months. The observations on the antioxidant enzymes in red blood cells are reported in Table 5. Amrita Bindu supplementation led to slow, progressive and appreciable increases in the activities of the antioxidant enzymes, SOD, GPx and CAT in the red blood cells and reached almost to the levels of healthy controls at the end of 6 months. A similar trend is seen in the blood free radical scavengers (Vitamins A, C, E and GSH) (Table 6). Six months of Amrita Bindu supplementation led

4. Discussion In the present investigation, 30 severe asthmatic children, 18 in the age group of 510 years and 12 in the age group

Table 6 Effect of Amrita Bindu supplementation on free radical scavengers in the blood of asthma children (mean S.D.) Vitamin A (mol/l) 510 years (n = 18) Initial 1 month 3 months 6 months 9 months 12 months Healthy controls 1118 years (n = 12) Initial 1 month 3 months 6 months 9 months 12 months Healthy controls

Vitamin C (mol/l) 34 36 42 50 58 62 4.0 3.5 4.3 5.1 3.5 4.2

Vitamin E (mol/l) 13.5 14.0 15.8 17.2 18.2 19.0 1.5 2.0 2.2 2.5 1.5 1.8

GSH (mol/l) 41 43 45 49 52 56 3.1 2.5 3.5 3.5 3.3 4.4

1.53 1.61 1.65 1.72 1.75 1.84

0.15 0.20 0.31 0.25 0.33 0.25

1.92 0.22 1.34 1.41 1.55 1.78 1.85 1.95 0.15 0.20 0.25 0.20 0.11 0.15

61 3.4 38 40 48 54 59 62 3 2 4 5 4 3

18.5 2.5 12.5 13.2 15.3 18.0 20.2 20.4 2.1 2.2 1.5 2.0 1.5 3.1

58 3.0 38 41 44 48 51 55 4.2 3.5 2.5 3.0 3.5 4.0

2.02 0.13

64 5

22.2 2.4

56 2.5

Statistically signicant differences from the initial level at the lowest month of supplementation: P < 0.05.

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105114

Superoxide radical scavenging property of Amrita Bindu in 1mM,3mM,5mM H 2O2
1400 Superoxide radical scavenging property of Amrita Bindu in 2mM,4mM,10mM H2O2
1600

111

1200

1400

1200

1000

1000

nanomoles of NBT

800

nanomoles of NBT 0 15 30 45
1mMwith AB 3mMwith AB 5mMwith AB

800

600

600

400
400

200
200

0 60
Time in Minutes

0 0 15 30 Time in Minutes 45 60

1mM without AB 3mMwithout AB 5mMwithout AB

2mM without AB 4mMwithout AB 10mMwithout AB

2mMwith AB 4mMwith AB 10mMwith AB

Fig. 1. Superoxide radical scavenging property of Amrita Bindu.

of 1118 years (who had asthma for 25 years in the former group and 712 years in the latter group), were on a 12-month period of Amrita Bindu supplementation. They were graded severe asthmatics according to the criteria of diagnosis of asthma (Farr, 1985). The lung capacity (PEFR) improved during supplementation and reached normal in 12 months (Table 3). The primary effect of Amrita Bindu appears to scavenge the free radicals (superoxide and hydroxyl radicals), reducing their levels in asthma to almost near normal levels at the end of 12 months for superoxide and 9 months for hydroxyl radical (Table 3). Earlier studies on rats with Amrita Bindu supplementation had not revealed any toxic effects on the organ systems. On the contrary, Amrita Bindu supplement provided protection against free radical and ROS induced tissue LPO, antioxidant depletion and the resultant tissue degeneration in rats challenged with free radical inducer

MNNG (Shanmugasundaram et al., 1994). Measurements on free radicals, LPO, antioxidant enzymes and free radical scavengers made on asthmatic children provided convincing proof concerning the effectiveness of Amrita Bindu supplementation as an inducer of antioxidants (signicant rise seen in SOD, GPx and CAT in Table 5) in children exposed to free radical challenge as in asthma. Amrita Bindu supplementation led to signicant reductions in TBARS levels, particularly in erythrocytes and RBC membranes (Table 4) and highly signicant improvements in the antioxidant enzyme proles are seen in these 30 asthma children (Table 5) possibly contributing relief to the respiratory distress. Free radical scavengers, Vitamin C and GSH were augmented in blood by Amrita Bindu (Table 6). This may be responsible for equipping them with a good antioxidant reserve to meet the oxidative challenge and keep the activities of other antioxidants also high and diminishing

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Hydroxyl radical scavenging property of Amrita Bindu in 1mM,3mM,5mM H2O2 Hydroxyl radical scavenging property of Amrita Bindu in 2mM,4mM,10mM H2O2

35

35

30

30

25

25

20 ng/ml H2O2 ng/ml H2O2 15

20

15

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0 0 15 30 45 Time in Minutes
1mMwith AB 3mMwith AB 5mMwith AB

0 60 0 15 30 45 Time in Minutes
2mM without AB 4mMwithout AB 10mMwithout AB

60

1mM without AB 3mMwithout AB 5mMwithout AB

2mMwith AB 4mMwith AB 10mMwith AB

Fig. 2. Hydroxyl radical scavenging property of Amrita Bindu.

the damage caused by the free radicals and lipid peroxides. These changes are associated with slow reduction of the severity and frequency of episodes of wheeze, the progressive reduction in the use of spacers/nebulisers and the eventual discontinuation of all drugs between 6 and 9 months of Amrita Bindu supplementation. It is a complex mixture of soluble and insoluble components. It can effectively prevent the formation of free radicals. This was tested by measuring the superoxide and hydroxyl radical concentrations formed in Fenton reaction mixture with and without Amrita Bindu. The incubation in Fenton reaction mixture (Figs. 1 and 2) is a classic system of free radical production, the superoxide and hydroxyl radicals. The free radical generation was inhibited by more than 60%. Amrita Bindu was able to suppress superoxide radical formation in different concentrations of hydrogen peroxide. At higher concentrations (>4 mM) of H2 O2 nearly 80% inhibition

was observed at 15 min duration suggesting that Amrita Bindu can efciently scavenge and intercept its formation (Fig. 1). Similarly more than 80% inhibition of hydroxyl radical concentration was seen in 5 mM H2 O2 (Fig. 2). The action of Amrita Bindu, shown in the Figs. 1 and 2, may be mediated through (a) adsorption of superoxide and/or hydroxyl radicals on the surface of the insoluble part of the supplement, or (b) adsorption of Fe2+ ions on the insoluble surface and inhibiting the Fenton reaction, or (c) by acting primarily on the hydrogen peroxide in the medium, or (d) by two or more of these mechanisms. Superoxide radical is measured in terms of NBT reduced, while hydroxyl radical is measured by its reaction with 2-deoxyribose. Abella et al. (1996) suggested a method to assess the total antioxidant and free radical scavenging property of a uid by incubating it with plasma and measuring the LPO products as TBARS. The total antioxidant property of Amrita Bindu

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105114

400

113

350

oxide, hydroxyl radicals formed excessively during asthma conditions are scavenged and intercepted, and thiol groups are maintained in the reduced state. This in turn, partially prevents loss of Vitamins C and E. Amrita Bindu act as an efcient antioxidant by scavenging the free radicals formed and controlling the LPO and tissue degeneration. 4.1. Antioxidant reinforcement by Amrita Bindu

300

250

200

150

Amrita Bindu used in our present study contained 8.0 mg Vitamin C, 4.2 mg Vitamin E and 9.45 mg sulphyhydryl (thiol) group per 500 mg powder. Though this is only a fraction of recommended dietary daily allowances of 40 mg Vitamin C and 30 mg Vitamin E in adults (Indian Council of Medical Research, 1992), it was still able to strengthen endogenous antioxidant defenses.

nmoles/dL

100

5. Conclusions The observations made by Amrita Bindu supplementation in the present study indicate that (a) Amrita Bindu supplement effectively improves the lung capacity, (b) Amrita Bindu reduces the oxidant damage, (c) Amrita Bindu acts as an antioxidant inducer-antioxidant enzymes are induced primarily by inactivating the free radicals formed in the system and secondly the presence of salt complexes quite possibly acting towards the replenishment of various antioxidants, particularly the enzymes SOD, GPx and CAT. Thirdly it may augment the protein thiol groups, replenishing the reducing power in the form of NADPH. This replenishment of NADPH as a result of recycling of GSSG into GSH may inuence several redox reactions in the cell and pave the way for the well known sparing vitamins on each other (Sen, 1995). However, studies on experimental animals with ligation of the segments of the gastro-intestinal tract can be an useful model in evaluating the free radical scavenging function of Amrita Bindu in vivo.

50

15

30

45

60

Time in Minutes

Controls with AB Asthma with AB

Controls Without AB Asthma Without AB

Fig. 3. Measure of overall antioxidant property of Amrita Bindu.

was determined as TBARS (Fig. 3). On standing, TBARS in plasma of normal or asthma patients increase by about 3.5%. This increase in plasma TBARS may be attributed to the action of oxidants in plasma, so the reduction of pre-existing LPO products in isolated plasma is intriguing. TBARS is a mixture of products, such as ethane to pentane, aldehydes including MDA, 4-hydroxy alkenals, 2-alkenals, 5-hydroxy octanol, butanone and many uorescent substances (Slater, 1984; Esterbauer et al., 1991; Siems et al., 1998). Amrita Bindu may have some reducing agents in solution which could reduce the aldehyde groups which may be acting on the pre-existing aldehydic LPO products in plasma and lowering the TBARS. Amrita Bindu is referred to as an antioxidant inducer because (a) the activity of antioxidant enzymes is increased (Table 5) and TBARS reduced (Table 4) when it is supplemented over a period of time, (b) the red cell GSH, blood ascorbic acid and plasma Vitamins A and E levels are raised (Table 6) without vitamin supplementation. It may be stated that by the induction of the antioxidant enzymes, super-

Acknowledgements The authors thank Dr. E.R.B. Shanmugasundaram M.A., B.Sc., Ph.D., F.R.I.C. (London), F.C.S. (London), Former Senior Professor, Head, University of Biochemistry Laboratories, Chennai, profusely for his wonderful gift of the test drug Amrita Bindu. The authors acknowledges the nancial help from CSIR in New Delhi, India, in the form of a Senior Research Fellowship.

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