Journal of Ethnopharmacology 90 (2004) 105–114

Amrita Bindu—an antioxidant inducer therapy in asthma childrenSumathy 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 theworld on the therapeutic use of antioxidants as supplements has revealed their capacity to control inflammatory processes.Amrita Bindu an Ayurvedic health food supplement has already shown to be an antioxidant inducer and to combat free radical-mediated tissuedamage studied in rats. Amrita Bindu is a salt–spice herbal mixture designed for positive health. It was tested as a supplement to therapy fora period of 12 months in 36 children suffering from asthma. Asthma is a chronic inflammatory disease with excessive free radical generationin lungs and blood cells. The patients were followed up by monitoring their clinical conditions, therapeutic doses of anti-asthmatic drugs, freeradical generation, lipid peroxidation (LPO) and antioxidants in blood. At the end of 3 months of Amrita Bindu supplementation, the patientshad 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; Inflammation; Lipid peroxidation

1. Introduction

Reactive oxygen species (ROS) can severely injure thelung. Lung is exposed to high concentrations of oxygen andits metabolites because of its large surface area (approxi-mately 100m2 in a 70 kg adult). ROS inhibit neutral en-dopeptidase activity in airway epithelium and aids in exag-geration of the bronchoconstrictor response to these peptide.In patients with asthma, oxidants present in the polluted air,exacerbate existing airway inflammation (Barnes, 1990).Evidence of excessive free radicals formation (superoxideand hydroxyl) and elevated levels of lipid peroxidation(LPO) has been observed in the red blood cells of asthmachildren (Shanmugasundaram et al., 2001). These free rad-icals are detoxified by primary intracellular antioxidant en-zyme systems comprising of superoxide dismutase (SOD),catalase activity (CAT), and glutathione peroxidase (GPx)(Sies and Stahl, 1995). Cross et al. (1994) have shown thatthe respiratory tract is lined with main antioxidants such asreduced glutathione (GSH), ascorbic acid, mucin, uric acid,metal binding proteins, antioxidant enzymes and sacrificialproteins. Smith et al. (1997), reported reduced SOD activityin the lung cells of asthma patients. Antioxidant deficiency

!Corresponding author. Tel.: +91-44-24480767;fax: +91-44-24926709.E-mail address: sunrays@md5.vsnl.net.in (K.R. Shanmugasundaram).

has been observed in the red blood cells of asthma chil-dren (Shanmugasundaram et al., 2001) which suggest thatdiminished antioxidant status served as a marker of inflam-mation characterising asthma. Alternatively, it may play arole in the development or severity of the disease. Manystudies all over the world showed that antioxidants servedas thrapeutic agents in controlling inflammatory responses(Romieu et al., 1998; Rust et al., 1998). One such antioxi-dant inducer therapy in our laboratory, aimed at augmentingnatural antioxidants is Amrita Bindu. It is a salt–spice herbalmixture, formulated based on the principles of Ayurvedaand siddha medicine. Indian medicine advocates the intakeof Jivanya (life sustaining) group of herbs and salts asfood additives. According to the C’araka and Samhita (1stcentury a.d.) Jivanya includes invigorators, nourishingdrugs, laxatives, wound healers and digestive stimulants.The second group is Balya or promoters of strength, com-plexion, voice and cardiac resilience to stress. The third areSowdhanyas, meaning cleansers of the organ systems. Theyused to prevent haemorrhoids, skin diseases, inflammationand infections. The fourth group of drugs clear the passagesin the mammary gland and the seminal tract and the fifthare used to promote secretions from eyes, nose and also forenemas. The sixth group includes antiemetics, and herbsthat control thirst, while the seventh include bowel binders,antidiuretics and diuretics. The eighth group of drugs arecuratives 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

106 S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114

is a set of pain relievers and the tenth group includes haema-tinics, sedatives and rejuvenators. In addition to commonsalt, Indian medicine advocates a variety of salts like rocksalt, saindhawa and bamboo salt that provides chlorides,phosphates and sulphates of potassium and magnesium andblack salt with sulphides and trace elements. Amrita Binduhas been formulated based on the above mentioned prin-ciples. It was found to combat free radical insults and toprovide protection against LPO and antioxidant depletionin nitrosamine-induced tissue damage studied in albino rats(Shanmugasundaram et al., 1994). Amrita Bindu has alsobeen tested as a supplement in diabetic retinopathy andhas been found to raise the antioxidant levels in blood andprevent further damage of the retina (Shanmugasundaramet al., 1996).The present study was undertaken to assess the effects of

Amrita Bindu supplementation in combating free radicalsand to act as an antioxidant inducer during asthma. Theefficiency of the Amrita Bindu in effectively scavenging freeradicals and its antioxidant potential were studied under invitro conditions.

2. Methodology

2.1. Subjects investigated

The studies were conducted on blood samples obtainedfrom asthma children in the age group of 5–18 years, whowere patients at Childs Trust Hospital, Nungambakkam,Chennai 600 034. They were mainly from middle income(socio-economic) groups and were under the therapeuticcare of Dr. Sarala Rajajee and Dr. Janani (Chief Paediatri-cians). Assessment of lung function tests, therapeutic mea-sures, clinical examinations were carried out by the medicaland paramedical staff in the centre.

2.2. Criteria for diagnosis of asthma

The children presented with wheezing, dyspnoea or diffi-cult breathing. They had experienced asthma episodes manytimes in the past, and were on medication. Every one of themhad a history of repeated respiratory infections, with wheez-ing heard when auscultating the chest. In some chronicsufferers with dyspnoea/difficult breathing, wheezing wasaudible after forced expiratory manoeuvre during pulmonaryfunction testing, with a prolonged expiratory phase suggest-ing obstruction in the airways. Evidence of upper airwaysounds, viscous and more tenacious sputum were the othersymptoms seen in this group to confirm asthma (Farr, 1985).Thirty-six patients having severe episodes of wheezing,

who often had regular attacks of asthma, with poor responseto usual bronchodilators and systemic steroids were involvedin our clinical trial to assess the therapeutic value of asalt–spice herbal preparation “AMRITA BINDU” as a healthfood supplement for a period of 12 months. Twenty-two chil-

dren (12 males,10 females) in the age group of 5–10 yearswere administered 250mg Amrita Bindu twice daily mixedwith 5ml honey after food. Fourteen children (8 males, 6females) of 11–18 years were administered 500mg AmritaBindu in hard gelatin capsules, twice daily after food. Theycontinued with all other medications. Informed consentswere obtained from the children and their parents and thestudy was approved by the Institutional Ethical Committee.Amrita Bindu is prepared by Prof. E.R.B. Shanmugasun-

daram, Herbal Research Foundation, Chennai. Amrita Binduis a salt–spice herbal mixture and the ingredients presentin it are (1) Induppoo or saindhawa, (2) Kalluppoo (rocksalt), (3) Moongiluppoo (pearl ash), (4) Valayaluppoo (ban-gle salt), (5) Karuuppoo (black salt), (6) Vengaram (crudeborax), (7) Tribulus terrestris L. (small caltrops), (8) Ca-latropis gigantean R.Br. (gigantic swallow wort and mu-dar), (9) Zingiber officinale (ginger), (10) Piper longumL. (dried catkins and long pepper), (11) Piper nigrum L.(black pepper), (12) Plumbago zeylanica L. (Ceylon lead-wort), (13) Cyperus rotundus L. (nutgrass). Its formula andits preparation is described in detail by Shanmugasundaramet al. (1994). Amrita Bindu is reported to have no hepato ornephrotoxicity when administered for long periods. Thirtypatients (18 in the age group of 5–10 years and 12 in theage group of 11–18 years) were followed up for a period of12 months. Blood analyses were made at the end of 1, 3,6, 9 and 12 months of Amrita Bindu supplementation. Fourpatients could not be followed up due to poor adherenceto medication and two did not come back to the hospital.There were six drop outs in the Amrita Bindu supplementedgroup. Fifty-two healthy children (32 children, 18 males, 14females in the age group of 5–10 years; and 20 adolescents,12 males, 8 females in the age group of 11–18 years) fromGovernment school, Nandanam, Chennai 600 035 were alsoincluded in the study as age and sex matched healthy con-trols (H.C.). None of the children smoked cigarettes or usedany form of tobacco. A standardised Child Health Question-naire (CHQ) was used to obtain all the information on res-piratory illness of the children and their parents, frequencyof spacers/inhalers used, drugs prescribed to children andon family background. Anthropometric measurements of allthe children were taken. The lung function tests (peak ex-piratory flow rate) were performed at the Childs Trust Hos-pital, Chennai, using Wright Peak Flowmeter. Each childblew into the Wright Peak Flowmeter three times and thehighest reading was accepted in each case. Repeat valueswere obtained from among one third of the volunteers ontwo different occasions, within 10 days. The variation wasless than 10%. All the subjects in this group continued withtheir usual diets and other activities. Medications were re-duced/discontinued depending on the clinical conditions atthe time of periodical monitoring. Records were maintainedon the dosage and frequency of the usage of anti-asthmaticdrugs. These children were examined by doctors who clas-sified as severe asthmatics in accordance with criteria fordiagnosis of asthma.

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114 107

2.3. Blood analyses

Fasting blood samples were drawn by venipuncture andaliquots were transferred to (a) tube containing EDTA(1mg/ml) for plasma and cell separation, (b) a tube con-taining 1mM diethyl dithiocarbamate (DDC) to inhibit theactivity of SOD and measure superoxide formation. Theblood samples were packed in ice during transportation tothe laboratory and analysis was initiated immediately.

2.4. Assays made in whole blood and plasma

Haemoglobin was assayed in whole blood by its oxida-tion to methaemoglobin with alkaline ferricyanide reagentgiving intensely coloured cyanmethaemoglobin. Blood glu-cose 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. Plasmawas separated (from blood samples collected with EDTAas anti-coagulant) by centrifugation at 4000 rpm for 10min.Vitamin A (Bayfield and Cole, 1980) and Vitamin E (Tayloret al., 1976) were assayed in plasma.

2.5. Antioxidant assays and lipid peroxidationmeasurement

The red cells were washed thrice with isotonic saline toremove the buffy coat and then sedimented. Cells were lysedwith ice cold water and membrane sedimented according tothe method of Dodge et al. with modifications reported ear-lier by Shanmugasundaram et al. (1992). SOD (Marklundand Marklund, 1974) and GPx (Rotruck et al., 1973) wereassayed in the haemolysate. CAT (Sinha, 1972) and mem-brane protein content (Lowry et al., 1951) were determinedin the cell membrane. The product of LPO was measured asTBARS and expressed in terms of malondialdehyde (MDA)in the plasma (Yagi, 1976), haemolysate and erythrocytemembrane (Cynamon et al., 1985).

2.6. Measurements of superoxide and hydroxyl freeradicals

Erythrocytes were lysed immediately, by mixing together0.5ml of cells and 10mM sodium phosphate buffer, pH7.4 containing 67mM NaCl, and 0.1mM EDTA. Thus, thehaemolysate prepared were divided into two aliquots to mea-sure superoxide and hydroxyl radicals. The superoxide rad-ical was determined in terms of the reduction of nitrobluetetrazolium (NBT) to formazan according to the method ofNishikimi et al. (1972). To one aliquot added 0.2ml of 4mMNBT containing 340mM sucrose and 2.0ml of 0.1M sodiumhydroxide in 24mM sodium hydrogen carbonate. The blueinsoluble formazan was dissolved in 5.0ml of 1,4-dioxanand read spectrophotometricaly. For each subject, one sam-ple of cells collected with SOD inhibited DDC, and onesample of cells without DDCwere processed simultaneously

and NBT reduction was measured. The difference betweenthe two is a measure of superoxide radical present at thattime.The hydroxyl radical in the haemolysate was measured by

the method of Gutteridge (1981) in which the hydroxyl rad-ical reacts with 2-deoxyribose. The resulting complex mix-ture of products was heated under acid conditions, and thedegradation products were measured as thiobarbituric acidreactive species (TBARS). 2.0ml of 20mM 2-deoxyribosewas added to the other aliquot and the mixture was incu-bated at 37 "C for 1 h. 0.5ml of thiobarbituric acid (TBA)and 0.5ml of trichloroacetic acid (TCA) were added andheated in a boiling water bath for 10–15min, cooled and readspectrophotometrically. Controls were included in which2-deoxyribose was omitted, and the residual thiobarbituricreactive acid species was measured as haemolysate. The dif-ference between the two readings gave the hydroxyl contentat that time.

2.7. Assessment of free radical scavenging property ofAmrita Bindu

Amrita Bindu was tested for its ability to neutralise su-peroxide and hydroxyl radicals formed in Fenton’s reagent.Fenton’s reagent was an aqueous solution of hydrogen per-oxide (1–10mM) containing Fe2+ at 30!M.Fenton’s reaction:

Fe2+ + H2O2 # Fe3+ + OH$ + OH•

Fe3+ + O2•$ # Fe2+ + O2

2.8. Superoxide and hydroxyl scavenging actionby Amrita Bindu

Superoxide and hydroxyl ions were generated in Fenton’smixture (Fe2+ + H2O2).Ten millilitres of incubation mixture were prepared by

adding 1ml of 300!M FeSO4 and 1ml of hydrogen per-oxide (10–100mM) to 8ml of 0.01M phosphate buffer atpH 7.4. In a duplicate set of tubes, 100mg of Amrita Bindususpended in the buffer was taken instead of buffer. Themixture was incubated at 37 "C for different time intervals(15, 30, 45 and 60min). To 4ml of aliquot, 0.2ml of NBTand 2.0ml of sodium hydroxide were added. The contentswere centrifuged and the precipitate was dissolved in 5ml of1,4-dioxan. The optical density was read at 520 nm. Each de-termination was duplicated. The superoxide radical presentin the incubation mixture and superoxide radical scavengingcapacity of Amrita Bindu were determined.In several tubes taken in duplicate 1ml of 300!M FeSO4

solution was mixed with 1ml of 0.01M sodium phosphatebuffer containing 20mM deoxyribose and 8ml of hydro-gen peroxide (10–100mM) in order to constitute 10ml ofthe reaction mixture. In one set of tubes, the buffer con-tained 100mg of Amrita Bindu in suspension. The mixturewas incubated at 37 "C for different time intervals (15, 30,

108 S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114

45 and 60min) and 1% TBA followed by 2.8% TCA wereadded. The contents were heated in a boiling water bath for10–15min, cooled and optical density was read at 532 nm.Hydroxyl radical generation and hydroxyl radical scaveng-ing capacity of Amrita Bindu were determined.

2.9. Assessment of the combined antioxidant and freeradical scavenging properties of Amrita Bindu

This study was designed to find out the free radicalquenching and combating capacity of Amrita Bindu byincubation with plasma and measuring the LPO productsfor the next 60min. Blood samples were obtained from 10asthma and 10 healthy children. The pooled plasma sampleswere taken as substrate. To 2.0ml of pooled plasma (asthmaand healthy children separately), was added 2ml of super-natant obtained by dissolving 100mg of Amrita Bindu inphosphate buffer and centrifuging. The mixture was incu-bated at 37 "C for different time intervals (0, 15, 30, 45 and60min). Phosphate buffer without Amrita Bindu was takenas a blank. The LPO levels were measured as TBARS.

2.10. Expression of cell and membrane constituents per1012 cells

The cell membrane constituents (catalase, LPO products)and free radical production (superoxide and hydroxyl radi-cals) are expressed per 1012 cells. This was arrived at fromthe cell count, haemoglobin in the lysate and membrane pro-tein measurements. From the total count and haemoglobinin lysate, the number of packed cells per millilitre were cal-culated.

2.11. Statistical analysis

The values are expressed as mean±S.D. for the asthmaticpatients and healthy population separately. Statistically sig-nificant differences was arrived at using student’s two-tailed‘t’ test.

Table 1General clinical and physical data on the population investigated (mean ± S.D.)

Parameters 5–10 years 11–18 years

Healthy controls (n = 18) Asthmatics (n = 18) Healthy controls (n = 12) Asthmatics (n = 12)

Age (years) 7 ± 3 7 ± 2 15 ± 4 15 ± 3Height (cm) 124 ± 7 118 ± 9 152 ± 8 144 ± 7Weight (kg) 22 ± 9 18 ± 7 43 ± 8 37 ± 6BMI (kg/m2) 14.3 ± 2.4 12.9 ± 2 18.6 ± 2.1 17.8 ± 2.3Duration of asthma (years) – 2–5 – 7–12Males (%) 63 61 45 33Percentage of patients using spacers/inhalers – 80 – 62Family historya of asthma (%) 9 55 14 58Family history of smoking (%) 30 70 21 64

a Atleast one parent having the disease.

3. Results

Table 1 shows the general data of 30 children who partic-ipated in the clinical trial of Amrita Bindu supplementationfor 12 months. These children were the regular out patientsof Childs Trust Hospital and were graded severe asthmaticsby the chief pediatrician. Twenty-two children were con-stantly using salbutamol nebuliser/inhaler (200!g per puff)atleast three times a day. Twelve children were taking be-clomethasone, daily doses of 400–650!g. In addition, 14children had asthalin, 2mg twice daily. Eight children in theage group of 11–18 years were taking theophyline, 10mg aday. These children were coughing, wheezing and had res-piratory distress over a period of 3–5 days a week. Theynever responded to cough expectorants and showed poorcompliance to these anti-asthmatic drugs. They were regu-larly missing their school days and attending the hospitalfor medical treatment. After a month, children respondedwell to the supplementation and started reducing their fre-quency 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 re-duced to occasional and these occasional episodes were verymild. Twenty-five children out of 30 became symptom freeafter 3 months and discontinued their usage of salbutamolnebulisers/spacers and bronchodilators. Their lung capacityalso improved (measured by Wright Peak Flowmeter). Theremaining five had reduced their salbutamol inhalation toonce a day (50!g per puff). All five had discarded salbuta-mol at the end of 9 months supplementation.Table 2 presents the effects of Amrita Bindu supplemen-

tation on general biochemical parameters in 30 asthmaticchildren (18 in 5–10 years and 12 in 11–18 years age group)parameters that were assayed to check for any adverse ef-fects. Blood haemoglobin levels were found to increasegradually by about 13% over a 12-month period of supple-mentation. Amrita Bindu supplementation did not produceany change in the blood glucose or non-protein nitrogen,indicating that it has no toxicity to the islets of langerhansand kidney.

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114 109

Table 2Effect of Amrita Bindu supplementation on biochemical parameters in severe asthmatics (mean ± S.D.)

Whole blood Serum

Hb (g/l) Glu (mmol/l) HbA1C (%) Urea (mmol/l) Uric acid (!mol/l) Creatinine (!mol/l)

5–10 years (n = 18)Initial 125 ± 4 4.9 ± 0.2 3.7 ± 0.3 3.4 ± 0.1 122 ± 7 36 ± 41 month 127 ± 3 4.8 ± 0.3 3.9 ± 0.2 3.5 ± 0.2 126 ± 5 37 ± 53 months 131 ± 4! 4.8 ± 0.4 4.1 ± 0.3 3.3 ± 0.3 131 ± 6! 37 ± 66 months 137 ± 3 4.4 ± 0.2! 3.7 ± 0.4 3.1 ± 0.3! 128 ± 9 35 ± 79 months 139 ± 5 4.5 ± 0.4 3.6 ± 0.2 3.1 ± 0.4 133 ± 8 36 ± 412 months 141 ± 2 4.2 ± 0.3 3.4 ± 0.3 3.1 ± 0.3 135 ± 9 35 ± 3

Healthy controls 136 ± 9 4.1 ± 0.4 3.9 ± 0.7 3.0 ± 0.2 127 ± 18 38 ± 7

11–18 years (n = 12)Initial 130 ± 6 5.6 ± 0.4 4.7 ± 0.2 3.7 ± 0.3 146 ± 14 39 ± 41 month 132 ± 5 5.5 ± 0.5 4.6 ± 0.3 3.6 ± 0.3 148 ± 12 38 ± 53 months 136 ± 7! 4.9 ± 0.6! 4.4 ± 0.2 3.5 ± 0.4 152 ± 15! 37 ± 66 months 140 ± 9 4.4 ± 0.7 4.5 ± 0.4 3.4 ± 0.2! 157 ± 16 39 ± 79 months 148 ± 6 4.6 ± 0.5 4.4 ± 0.4 3.4 ± 0.4 163 ± 11 38 ± 412 months 151 ± 4 4.3 ± 0.4 4.3 ± 0.2 3.2 ± 0.3 172 ± 13 39 ± 5

Healthy controls 145 ± 11 4.7 ± 0.6 4.3 ± 0.6 3.3 ± 0.3 167 ± 30 43 ± 9! Statistically significant differences from the initial level at the lowest month of supplementation: P < 0.05.

Amrita Bindu supplementation raised the lung capacity asmeasured by peak expiratory flow rate to about 65% almostreaching the levels of controls (Table 3 measured at theintervals of 6 months).Table 3 gives the level of superoxide and hydroxyl radi-

cals formation in the red blood cells in children during sup-plementation. Significant decreases in the level of both freeradicals were noticed after 3 months of supplementation,while superoxide radical generation reached a normal level(compared to healthy subjects) in 12 months, hydroxyl radi-

Table 3Effect of Amrita Bindu supplementation on free radical formation in redblood cells, and PEFR of asthma children (mean ± S.D.)

Superoxide(mmolNBT/1012cells)

Hydroxyl (nmolMDA/1012 cells)

PEFR(l/min)

5–10 years (n = 18)Initial 108 ± 7 14 ± 2 108 ± 211 month 97 ± 4 12 ± 3 –3 months 74 ± 5! 8 ± 2! –6 months 58 ± 3 5 ± 2 136 ± 30!

9 months 44 ± 4 3 ± 1.5 –12 months 32 ± 6 3 ± 1 178 ± 36

Healthy controls 38 ± 8 3 ± 1.5 235 ± 37

11–18 years (n = 12)Initial 108 ± 7 27 ± 3 201 ± 181 month 97 ± 4 24 ± 2 –3 months 74 ± 5! 18 ± 3! –6 months 58 ± 3 11 ± 4 247 ± 33!

9 months 44 ± 4 8 ± 3 –12 months 32 ± 6 4 ± 2.5 286 ± 26

Healthy controls 38 ± 8 4.6 ± 2 263 ± 47! Statistically significant differences from the initial level at the lowest

month of supplementation: P < 0.05.

cal generation reached normal levels at the end of 9 monthstherapy.Table 4 shows the LPO levels in the blood of asthmatic

children treated with Amrita Bindu for a period of 12months. It was observed that supplementation of AmritaBindu led to a steady decrease in the levels of LPO productin plasma, RBC lysate and RBC membranes. LPO prod-ucts in plasma had decreased by 48% and had reached thecontrol values after 9 months of Amrita Bindu supplemen-tation. Reduction in the TBARS in the haemolysate as wellas in the membrane fraction were much greater after Amrita

Table 4Effect of Amrita Bindu supplementation on LPO products (TBARS)expressed as MDA in blood of asthma children (mean ± S.D.)

Plasma(nmol/dl)

Cell lysate(nmol/1012 cells)

Membrane(nmol/1012 cells)

5–10 years (n = 18)Initial 328 ± 15 26 ± 2 546 ± 841 month 312 ± 20 24 ± 2.5 522 ± 683 months 276 ± 22! 21 ± 1.5! 487 ± 75!

6 months 232 ± 19 16 ± 2.0 402 ± 539 months 197 ± 21 13 ± 1.8 342 ± 6212 months 172 ± 15 10 ± 1.5 285 ± 46

Healthy controls 163 ± 16 9 ± 2.0 235 ± 37

11–18 years (n = 12)Initial 342 ± 21 29.6 ± 3 656 ± 961 month 320 ± 20 27 ± 3.5 642 ± 553 months 280 ± 28! 25 ± 3.5! 587 ± 62!

6 months 235 ± 22 21 ± 2.2 498 ± 719 months 200 ± 25 18 ± 2 405 ± 5712 months 174 ± 18 13 ± 1.5 325 ± 66

Healthy controls 171 ± 20 10.5 ± 1.5 242 ± 28! Statistically significant differences from the initial level at the lowest

month of supplementation: P < 0.05.

110 S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114

Table 5Effect of Amrita Bindu supplementation on antioxidant enzymes in bloodof asthma children (mean ± S.D.)

SOD(IU/mgHb)

GPx(!gGSH/mgHb/min)

CAT(mmolH2O2/1012cells/min)

5–10 years (n = 18)Initial 1.94 ± 0.26 5.1 ± 0.3 3.9 ± 0.201 month 2.11 ± 0.32 5.4 ± 0.4 4.1 ± 0.223 months 2.80 ± 0.23! 5.7 ± 0.2! 4.5 ± 0.3!

6 months 3.42 ± 0.21 6.0 ± 0.3 4.6 ± 0.259 months 3.55 ± 0.15 6.3 ± 0.3 4.8 ± 0.3012 months 3.61 ± 0.20 6.5 ± 0.4 4.9 ± 0.28

Healthy controls 3.64 ± 0.13 6.66 ± 0.3 4.85 ± 0.31

11–18 years (n = 12)Initial 1.71 ± 0.24 4.6 ± 0.4 3.3 ± 0.311 month 1.92 ± 0.22 4.8 ± 0.3 3.4 ± 0.423 months 2.42 ± 0.3! 5.2 ± 0.2! 3.9 ± 0.21!

6 months 2.91 ± 0.25 5.8 ± 0.3 4.2 ± 0.339 months 3.30 ± 0.30 6.1 ± 0.3 4.4 ± 0.1512 months 3.46 ± 0.22 6.4 ± 0.2 4.5 ± 0.23

Healthy controls 3.55 ± 0.13 6.3 ± 0.3 4.51 ± 0.26! Statistically significant differences from the initial level at the lowest

month of supplementation: P < 0.05.

Bindu supplementation, they were reduced almost to halfthe initial high value at the end of 1-year period of AmritaBindu supplementation. Statistically significant decrease inthe 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 supplementationled to slow, progressive and appreciable increases in theactivities of the antioxidant enzymes, SOD, GPx and CAT inthe red blood cells and reached almost to the levels of healthycontrols at the end of 6 months. A similar trend is seen in theblood free radical scavengers (Vitamins A, C, E and GSH)(Table 6). Six months of Amrita Bindu supplementation led

Table 6Effect of Amrita Bindu supplementation on free radical scavengers in the blood of asthma children (mean ± S.D.)

Vitamin A (!mol/l) Vitamin C (!mol/l) Vitamin E (!mol/l) GSH (!mol/l)

5–10 years (n = 18)Initial 1.53 ± 0.15 34 ± 4.0 13.5 ± 1.5 41 ± 3.11 month 1.61 ± 0.20 36 ± 3.5 14.0 ± 2.0 43 ± 2.53 months 1.65 ± 0.31 42 ± 4.3! 15.8 ± 2.2! 45 ± 3.5!

6 months 1.72 ± 0.25! 50 ± 5.1 17.2 ± 2.5 49 ± 3.59 months 1.75 ± 0.33 58 ± 3.5 18.2 ± 1.5 52 ± 3.312 months 1.84 ± 0.25 62 ± 4.2 19.0 ± 1.8 56 ± 4.4

Healthy controls 1.92 ± 0.22 61 ± 3.4 18.5 ± 2.5 58 ± 3.0

11–18 years (n = 12)Initial 1.34 ± 0.15 38 ± 3 12.5 ± 2.1 38 ± 4.21 month 1.41 ± 0.20 40 ± 2 13.2 ± 2.2 41 ± 3.53 months 1.55 ± 0.25 48 ± 4! 15.3 ± 1.5! 44 ± 2.5!

6 months 1.78 ± 0.20! 54 ± 5 18.0 ± 2.0 48 ± 3.09 months 1.85 ± 0.11 59 ± 4 20.2 ± 1.5 51 ± 3.512 months 1.95 ± 0.15 62 ± 3 20.4 ± 3.1 55 ± 4.0

Healthy controls 2.02 ± 0.13 64 ± 5 22.2 ± 2.4 56 ± 2.5!

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

to rise in the Vitamins A, C, E and GSH levels compared tothose seen in healthy age matched children.

3.1. Assessment of free radical scavenging and totalantioxidant property of Amrita Bindu

Amrita Bindu was tested for its ability to scavenge super-oxide and hydroxyl radicals formed in the Fenton reaction.Amrita Bindu added to the reaction mixture was able tolower the formation of superoxide radical. The percentageinhibition of formation of superoxide radical was more than63%, and the effect was more pronounced at 15min whenwhole of Amrita Bindu was used (Fig. 1). Similar observa-tions were seen with hydroxyl formation in Fenton reactionwith and without Amrita Bindu (Fig. 2). These observationsindicate that Amrita Bindu can effectively scavenge andintercept the free radicals, superoxide and hydroxyl radi-cals formed in the reaction mixture. The overall antioxidantproperty of Amrita Bindu was also assessed by measuringthe LPO products over 60min (Fig. 3). Pooled plasma ob-tained from 10 healthy children and 10 asthmatic children,served as the substrate. LPO measured as TBARS showed amarginal decrease in healthy samples, whereas decreases inthe order of 40 nmol/dl were noted in asthma samples after60min with Amrita Bindu. In the case of blanks withoutAmrita Bindu, LPO levels increased after 1 h. These resultsshow that Amrita Bindu act as a free radical quencher andcan combat the free radicals as and when they are formedin this system.

4. Discussion

In the present investigation, 30 severe asthmatic children,18 in the age group of 5–10 years and 12 in the age group

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Superoxide radical scavenging property of Amrita Bindu in

1mM,3mM,5mM H 2O2

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1mM without AB 1mMwith AB3mMwithout AB 3mMwith AB5mMwithout AB 5mMwith AB

Superoxide radical scavenging propertyof Amrita Bindu in 2mM,4mM,10mM

H2O2

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200

400

600

800

1000

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1400

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0 15 30 45 6 0Time in Minutes

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

of 11–18 years (who had asthma for 2–5 years in the for-mer group and 7–12 years in the latter group), were on a12-month period of Amrita Bindu supplementation. Theywere graded “severe asthmatics” according to the criteria ofdiagnosis of asthma (Farr, 1985).The lung capacity (PEFR) improved during supplemen-

tation and reached normal in 12 months (Table 3). The pri-mary effect of Amrita Bindu appears to scavenge the freeradicals (superoxide and hydroxyl radicals), reducing theirlevels in asthma to almost near normal levels at the end of12 months for superoxide and 9 months for hydroxyl rad-ical (Table 3). Earlier studies on rats with Amrita Bindusupplementation had not revealed any toxic effects on theorgan systems. On the contrary, Amrita Bindu supplementprovided protection against free radical and ROS inducedtissue LPO, antioxidant depletion and the resultant tissuedegeneration in rats challenged with free radical inducer

MNNG (Shanmugasundaram et al., 1994). Measurementson free radicals, LPO, antioxidant enzymes and free radicalscavengers made on asthmatic children provided convinc-ing proof concerning the effectiveness of Amrita Bindu sup-plementation as an inducer of antioxidants (significant riseseen in SOD, GPx and CAT in Table 5) in children exposedto free radical challenge as in asthma. Amrita Bindu sup-plementation led to significant reductions in TBARS levels,particularly in erythrocytes and RBC membranes (Table 4)and highly significant improvements in the antioxidant en-zyme profiles are seen in these 30 asthma children (Table 5)possibly contributing relief to the respiratory distress.Free radical scavengers, Vitamin C and GSH were aug-

mented in blood by Amrita Bindu (Table 6). This maybe responsible for equipping them with a good antioxi-dant reserve to meet the oxidative challenge and keep theactivities of other antioxidants also high and diminishing

112 S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114

Hydroxyl radical scavenging property ofAmrita Bindu in 1mM,3mM,5mM H2O2

0

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35

0 15 30 45 60Time in Minutes

ng/m

l H2O

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Hydroxyl radical scavenging property ofAmrita Bindu in 2mM,4mM,10mM H2O2

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

the damage caused by the free radicals and lipid perox-ides. These changes are associated with slow reductionof the severity and frequency of episodes of wheeze, theprogressive reduction in the use of spacers/nebulisers andthe eventual discontinuation of all drugs between 6 and 9months of Amrita Bindu supplementation. It is a complexmixture of soluble and insoluble components. It can ef-fectively prevent the formation of free radicals. This wastested by measuring the superoxide and hydroxyl radicalconcentrations formed in Fenton reaction mixture with andwithout Amrita Bindu. The incubation in Fenton reactionmixture (Figs. 1 and 2) is a classic system of free radicalproduction, the superoxide and hydroxyl radicals. The freeradical generation was inhibited by more than 60%. AmritaBindu was able to suppress superoxide radical formationin different concentrations of hydrogen peroxide. At higherconcentrations (>4mM) of H2O2 nearly 80% inhibition

was observed at 15min duration suggesting that AmritaBindu can efficiently scavenge and intercept its formation(Fig. 1). Similarly more than 80% inhibition of hydroxylradical concentration was seen in 5mM H2O2 (Fig. 2). Theaction of Amrita Bindu, shown in the Figs. 1 and 2, maybe mediated through (a) adsorption of superoxide and/orhydroxyl radicals on the surface of the insoluble part of thesupplement, or (b) adsorption of Fe2+ ions on the insolublesurface and inhibiting the Fenton reaction, or (c) by actingprimarily on the hydrogen peroxide in the medium, or (d)by two or more of these mechanisms. Superoxide radical ismeasured in terms of NBT reduced, while hydroxyl radicalis 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 fluid byincubating it with plasma and measuring the LPO productsas TBARS. The total antioxidant property of Amrita Bindu

S.S. Kumar, K.R. Shanmugasundaram / Journal of Ethnopharmacology 90 (2004) 105–114 113

0

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Asthma with AB Asthma Without AB

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

was determined as TBARS (Fig. 3). On standing, TBARS inplasma of normal or asthma patients increase by about 3.5%.This increase in plasma TBARS may be attributed to the ac-tion of oxidants in plasma, so the reduction of pre-existingLPO products in isolated plasma is intriguing. TBARS is amixture of products, such as ethane to pentane, aldehydesincluding MDA, 4-hydroxy alkenals, 2-alkenals, 5-hydroxyoctanol, butanone and many fluorescent substances (Slater,1984; Esterbauer et al., 1991; Siems et al., 1998). AmritaBindu may have some reducing agents in solution whichcould reduce the aldehyde groups which may be acting onthe pre-existing aldehydic LPO products in plasma and low-ering the TBARS.Amrita Bindu is referred to as an antioxidant inducer be-

cause (a) the activity of antioxidant enzymes is increased(Table 5) and TBARS reduced (Table 4) when it is supple-mented over a period of time, (b) the red cell GSH, bloodascorbic acid and plasma Vitamins A and E levels are raised(Table 6) without vitamin supplementation. It may be statedthat by the induction of the antioxidant enzymes, super-

oxide, hydroxyl radicals formed excessively during asthmaconditions are scavenged and intercepted, and thiol groupsare maintained in the reduced state. This in turn, partiallyprevents loss of Vitamins C and E. Amrita Bindu act as anefficient antioxidant by scavenging the free radicals formedand controlling the LPO and tissue degeneration.

4.1. Antioxidant reinforcement by Amrita Bindu

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

5. Conclusions

The observations made by Amrita Bindu supplementa-tion in the present study indicate that (a) Amrita Bindusupplement effectively improves the lung capacity, (b) Am-rita Bindu reduces the oxidant damage, (c) Amrita Binduacts as an antioxidant inducer-antioxidant enzymes are in-duced primarily by inactivating the free radicals formedin the system and secondly the presence of salt complexesquite possibly acting towards the replenishment of variousantioxidants, particularly the enzymes SOD, GPx and CAT.Thirdly it may augment the protein thiol groups, replen-ishing the reducing power in the form of NADPH. Thisreplenishment of NADPH as a result of recycling of GSSGinto GSH may influence several redox reactions in the celland pave the way for the well known sparing vitamins oneach other (Sen, 1995).However, studies on experimental animals with ligation

of the segments of the gastro-intestinal tract can be an usefulmodel in evaluating the free radical scavenging function ofAmrita Bindu in vivo.

Acknowledgements

The authors thank Dr. E.R.B. Shanmugasundaram M.A.,B.Sc., Ph.D., F.R.I.C. (London), F.C.S. (London), FormerSenior Professor, Head, University of Biochemistry Labora-tories, Chennai, profusely for his wonderful gift of the testdrug Amrita Bindu. The authors acknowledges the financialhelp from CSIR in New Delhi, India, in the form of a SeniorResearch Fellowship.

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