Ind ian Journ al of Experimental Biology VoL 39, May 200 I, pp. 447-452

Antioxidant activity of brahma rasayana

Rekha P S, Girija Kuttan & Ramadasan Kuttan*

A mala Cancer Research Centre, A mala Nagar P 0, Thrissur, 680553, India.

Received I I April 2000: revised 6 Febmary 2001

Free oxygen rad ical scavenging activity of brahma rasayana(BR) was studied by in vitro and in vivo model s. Addition of aqueous extract of BR was found to scavenge the lipid peroxides already present in rat liver homogenate(IC50 700~g/ml) and inhibit the lipid peroxide generated by Fe2

+- ascorbate(IC50 2600~g/m l ) and Fe3+- ADP - ascorbate system(IC50

1 200~g/ml ). BR was found to scavenge the hydroxyl radical generated by Fenton reaction (IC50 7400~g/ml) and superoxide generated by photoreduction of ribonavin (IC50 1 80~g/ml ). BR was also found to inhibit the nitric ox ide radical generated in vitro from sodium nitroprusside (IC50 5 .5~g/m l ). Oral administrat ion of BR(50mg/dosc/animal) was found to inhibit the PMA induced superoxide generation in mice peritoneal macrophages. Oral administration of BR ; 10 and 50mg/dosc/animal was also found to inhibit the nitrite production in peritoneal macrophages and percentage inhibition was 25.2% and 37.89r respectively. These results indicate significant an tioxidant activity of BR in vitro and in vivo.

Reacti ve oxygen spec ies(ROS) and free rad ica ls which are fo rmed in the body as a consequence of normal metabolic reacti ons, ex posure to ioni s ing rad iation and by the in fl ue nce of many xenobiot ics are indicated in the ca usati on of several di seases. Ant ioxidants, which can scavenge free radi cals have an important ro le in biological syste m and the ir use is implicated in the preventi on of cancer, heart di seases, ag ing etc. Human body has an inhere nt mechanism to reduce the free rad ica ls induced injury by enzy mat ic or non-enzymati c methods 1• When the normal level of antiox idant defense mechani sm is not suffi c ient for the eradicat ion of free radica ls induced inj ury, admini strati on of antioxidants have a protecti ve ro le to play. Several antiox idants of plant ori g in are experimentall y proved and used as effecti ve protec ti ve agents aga inst ox idati ve stress2

.

Indi genous medic ines in Indi a have several preparati ons which are implicated in preventi ve medic ine. Rasayanas are a group of non-tox ic po lyherbal drug preparation, which are immunostimulatory and thereby prevent the causati on of the di seases3

. W e have earlier reported that rasayanas could reduce the side effects of radiati on and chemotherap/·5 and could stimul ate the immune ce ll s6

'7

. It was al so fo und to significantly reduce the methylcholanthrene induced sarcoma in mice8 and transpl anted tumors in anima ls9

• In the present study

*Correspondent author : Fax : 0091487 211 020.E-mail : di rector @amala.com

we have evaluated the pote ncy of brahma rasayana as an antiox idant.

Materials and Methods Brahma rasayana(BR) was purchased from

Va idyaratnam Oushadhasa la, Ollur, India (composi tion of BR and mode of preparation is g iven in Table 1). Ni troblue tetrazolium (NBT), adenosi ne diphosphate (ADP) and N(1-naphthylethylenediamine d ihydro­chloride were purchased from Sisco Research Laboratories Pvt. Ltd, Bombay. 2-deoxy-D-ri bose was obtained fro m Sigma Chemica l Co. St. Louis M.O. Phorbol 12- myristate- 13-acetate (PMA) was a gift from Dr. Allan Canney, USA. T issue culture medi um RPMI-1 640 was obtained fro m Hi -media Laboratories, Bombay. Foetal calf serum(FCS) was obtained from Biological Industries, Kibbutz Beit Haemek, Israel. All other chemicals and reagents used were of analytical grade. Aqueous extract of BR was prepared by freshly stirring BR in water for lhr and centrifuged and the supernatant was used for the assay.

Inbred strains of Balb/c mice(20-25g, 4-S weeks o ld) were purchased from National Centre for Laboratory Animal Sciences, Hyderabad. They were housed in ventilated cages in air controlled rooms and fed with normal mouse chow (Lipton , Indi a) and water ad libitum.

Determination of antioxidant activity of brahma rasayana in vitro

Effect of BR on inhibition of lipid peroxide fo rmation induced by Fi+ -ascorbate system -

448 INDIAN J EXP BIOL., MAY 2001

Reaction mixture (0.5ml) contammg 25 % rat liver homogenate (O.lml) w/v in tris-HCI buffer(40mM, pH 7.0), potassium ch loride(30mM), ferrous iron(0.16mM) and ascorbic acid(0.06mM) was incubated for 1hr at 37°C in presence and absence of different concentrations of BR. The lipid peroxide formed was measured by the method of Ohkawa et nl 10

. For this 0.4ml of reaction mixture was treated with sodium dodecyl sulphate (SDS-0.2ml,8.1 %), thiobarbituric acid(TBA-1.5mi,0.8%) and acetic acid ( 1.5ml,2.5% of pH 3.5). The mixture(4ml) was then kept in a water bath at 95°C for lhr. After cooling, lml of distilled water and 5ml of a mixture of n­butanol and pyridine(l5: 1 v/v) were added and shaken vigorously. After centrifugation, the chromophore was measured at 532nm. The percentage inhibition of lipid peroxidation was determined by comparing the result of control and test compounds.

Effect of BR 011 inhibition of lipid peroxide formation induced by Fe3

+ -ADP-ascorbate system-

The incubation mixture contained 10% rat liver homogenate (0.5ml), fetTic i on (0.1 mM), ADP( l .7mM), ascorbic acid(0.5mM) and the final volume was made up to 1.5m1 with KCI (0.15M). Mixture was incubated for 20min at 37°C in presence and absence of different concentration of BR. After incubation 0.6ml of reaction mixture was taken and inhibition of lipid peroxidation was determined by estimation of thiobarbituric acid reacting substances (TBARS) as described by Ohkawa et al. 10 as given above.

Effect of BR on hydroxyl radical scavenging activity-Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compounds for ' ydroxyl radical generated by Fe2+-ascorbate-EDT A-H20 2 (Fenton reaction 11

). The hydroxyl radical s attack deoxyribose that eventually result in TBARS formation.

The reaction mi xture contained deoxyribose(2.8mM), FeCI3(0.1 mM), EDT A(0.1 mM),

Table 1- Composition or brahma rasayana(BR) and mode of preparation 19

I . Emblica offlcinalis(20%) 19. Desmostachya bipinnata(0.4%)

2. Terminalia chebula(6.67%) 20. Saccharum offlcinarum(0.4 o/t )

3. Urarira pitca(0.4%) 21. 01yza malampu:hensis(0.4% )

4. Desmodium gangeticum(0.4%) 22. Ci1liWIIIOmu111 inn·s(0 . 16%)

5. Cmelina arborea(0.4%) 23. Elellaria cardamomU11 1(0 . 16' c)

6. Solanumnigrum(0.4%) 24. Cypems rotundus(O.I6% )

7. Tribulus terrestris(0.4%) 25. Curcuma /onga(O.I6 %)

8. 1\eg/e marme/os(0.4%) 26. Piper long um(0 . 16%)

9. Premna tomentosa(0 .4%) 27. !\qui/aria aga//ocha(O. I6% )

I 0. StereospemiUIII suvaeolens(0.4%) 28. Santalum albwn(O. I6%)

II . Sida rhombilfolia(0.4%) 29. Cente//a asciatica(0.16%)

12. Boerhaavia diffusa(0.4%) 30. Me sua f errea(0.16%)

13 . Ricinus CO III111111lis(0.4%) 31 . Clitoria temata(0. 16%)

14. Vigna vexilata(0.4%) 32. Acarus calamus(0. 16%)

15 . Phaseolus adenanthus(0.4%) 33. Scirpus crossus(O.l6o/o)

16. Asperagus racemosus(0.4%) 34. Clycyrrhiza glabra(0.16 '7o )

17. Holostemma annulare(0.4%) 35. Embelia ribes(0. 16%)

18. Leptadenia reticulata(0.4%)

Items I -21 were made into small pieces and washed it well. 16part of water was added to the total quantity or drugs and allowed to boil to get one foUith of original volume. The seeds from fruit of Emblica oJ]icinalis and Terminalia chebu/a were removed and the pulp was roasted well by adding sufficient quantity of ghec and sesame oil. Then sufficient quantity of sugar was added to the above decoction to get a paste like formation. The plants 22-35 were cleaned and dried well and was made into fine powder and this powder was mixed with above paste and stirred well. When the preparation comes to normal temperature, sufficient quantity of honey was added. mixed well and stored at room temperature.

REKHA eta/.: ANTIOXIDANT ACTIVITY OF BRAHM A RASA YANA 449

H20 2( 1 mM), ascorbic acid(0.1 mM), KH2P04-KOH buffer(20mM, pH 7.4) and various concentrations of BR in a final volume of 1ml. The reaction mixture was incubated for l hr at 37°C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al 10

. The percentage inhibition was determined by comparing the results of test compounds and control.

Effect of BR on inhibition of superoxide radical generation-Superoxide scavenging was determined by the NBT reduction method of McCord and Fridovich12. The reaction mixture contained EDTA(6.6mM) containing 3j..lg NaCN, riboflavin(2j..lM), NBT(50j..lM), various concentrations of BRand phosphate buffer (67mM, pH 7.8) in a final volume of 3ml. The tubes were uniformly illuminated with an incandescent lamp for l5min . and the optical density was measured at 530nm before and after the illumination. The percentage inhibition of superoxide generation was measured by comparing the absorbance values of control and those of test compounds.

Effect of BR on inhibition of nitric oxide radical generation-Aqueous solution of sodium nitroprusside spontaneously generates nitric oxide (NO) at physiological pH, which interacts with oxygen to produce nitrite ions and which was measured colorimetrically 13'14. 3ml of reaction mixture containing sodium nitroprusside(lOmM) in phosphate buffered saline(PBS) and various concentrations of water extract of BR was incubated at 25°C for 150min. Controls without test compound was kept in an identical manner. After incubation 0.5ml of reaction mixture was removed and 0.5ml of Griess reagent (1% sulphanilamide, 2% H3P04 and 0.1% naphthylethylenediamine dihydrochloride) was added. The absorbance of the chromophore formed was read at 546nm. The percentage inhibition of nitric oxide generation was measured by comparing the absorbance values of control and those of test compounds.

Effect of BR on nitrite production in peritoneal macrophages-Nitric oxide(NO) produced by macrophages quickly reacts with oxygen to produce nitrite ions. The nitrite concentration in the cell free culture supernatant of peritoneal macrophages were measured spectrophotometrically 15'16. Inbred strains of female Balb/c mice(20-25g, 4-5 weeks old) were used for the experiment. Peritoneal macrophages were elicited in all animals by injecting a 5% solution of sodium caesinate (0.2ml) intraperitoneally. Five days

after injection , macrophages were harvested by peritoneal lavage using sterile phosphate buffered saline(PBS). Cells were washed, centrifuged and l x 106 cells were cultured in 96 well flat bottom titre plates in RPMI-1640 supp lemented with 5% FCS for 24hr at 37°C in presence and absence of various concentrations of methanolic extract of BR. Culture plates were centrifuged and medium from five wells of treated and untreated wells were pooled. 1 ml of medium (in triplicate) was mixed with sulphosalicylic acid (0.1 ml, 70% ), vortexed, centrifuged and the supernatant was mixed with 5% NH4CI solution (0.8ml) containing sodium borate buffer pH9, NaOH (0.2ml, 10%) and Griess reagent (0.5ml). The reaction mixture was incubated at 60°C for lOmin and at 4°C for 5min. The optical density was measured at 546nm, which is a measure of the amount of nitrite ions produced by the peritoneal macrophages. In all the experiments the nitrite content in the well containing medium without cells alone and with drug were determined as controls and substracted from the corresponding normal values.

Determination of antioxidant activity of brahma rasayana in vivo

Effect of BR on nitrite production in peritoneal macrophages-lnbred strains of female Balb/c mice(20-25g, 4-5weeks old) were used for this study. They were divided into three groups (3animals/group). Peritoneal macrophages were elicited in all animals by injecting a 5% solution of sodium caesinate (0.2ml) intraperitoneally. Group I served as untreated control. Group II was treated with daily single dose of BR(10mg/animal for 5 days, po). Groupiii was treated with daily single dose of BR (50mg/animal, po) for 5 days. Peritoneal macrophages were harvested on fifth day after drug administration and cells were cultured (lx106 cells/well/0.25ml) in 96 well flat titre plates in RPMI -1640 supplemented with 5% FCS for 24hr. The concentration of nitrite ions formed in the cell free culture supernatant was determined by the method of Ding et al. 15 as given above.

Effect of BR on PMA induced superoxide generation in peritoneal nzacrophages-Inbred strains of female Balb/c mice(20-25g, 4-5 weeks old) were divided into three groups(3animals/group). All the animals were injected(ip) with 0.2ml of sodium caesinate(5%) to elicit macrophages. Group I kept as untreated control. Group II was treated with daily single dose of BR(50mg/animal for 5 days, po).

450 INDIAN J EX P BIOL. , MAY 2001

Group III was treated with single dose of BR(50mg/animal on day 1). Peritoneal macrophages elicited by sodium caesinate were activated in vivo on 51

h day by injecting PMA (100 ng/animal , ip). Three hour after activation, peritonea l macrophages were harvested. The effect of the test compounds on the inh ibition of superox ide generation m the macrophages were meas ured by inhibition in the reduction of BT to formaza n by the method of Dwivedi et al 11

. The reaction mixture ( l .Oml ) contained in a ratio of 6:2: 1 of NBT (0.2% in PBS, pH 7.4), dextrose(5%) and Hank's balanced sa lt solution(pH 7.4) was mixed with 0.5ml of peritonea l macrophages (l x 106 cells/ml) and incubated for 45minutes at room temperature. The mixture was centrifuged and cell pellet was boiled with 2ml of pyridine for 10minute. The opt ical density of the supernatant was measured at 515 nm, which is a measure of the superox ides produced by the activated peritonea l macrophages. The percentage inhibition was determined by comparing the absorbance values of untreated and treated animals.

Results

In vitro study Inhibition of lipid peroxidation by BR- Addition

of BR was found to inhibit perox ides generated by Fe2+-ascorbate and Fe3+-ADP-ascorbate in rat li ver homogenate (Fig. 1A). The concen tration of BR needed for 50% inhibition was found to 2600 j..tg/ml , 1200j..tg/ml respecti vely. Addition of BR was also found to scavenge lipid peroxides already present and the concentration needed for 50% inhibition was fou nd to be 700 j..tg/ml(Fig lA). Time course of lipid peroxidati on induced by Fe2+-ascorbate as shown by TBARS in the absence and presence of BR(5mg/ml) is shown in Fig. lB .

Inhibition of hydro.xyl radical by BR-Degradation of deoxyribose by hydroxyl radical generated from Fe3+-ascorbate-EDTA-H20 2 system was found to be inhibited by BR. Concentration of BR needed for 50% inhibition was found to be 7400 j..tg/ml (Fig. 2A).

Inhibition of superoxide radical by BR- BR was found to scavenge the superox ide radical generated by photoreduction of ribofl av in . Concentration of BR needed for 50% inhibition was found to be 180 j..tg/ml (Fig 2B).

Inhibition of nitric oxide radical by BR - Nitric ox ide radical generated from sodium nitroprusside at physiological pH was found to be inhibited by BR.

-· • --- Ferrous (Fe2+) · ascorbate ... X - .. Ferric (Fe:>+) • ADP- ascorbate

§ A --· • -- Without any induction

~ ::te?:::::;;:~===~·:::::;~-0 1 2 3 4

0·9 B -· o -- Wilhou1 BR

E o.a ... x--- With SA c N (")

lO 0 .6

"' Q)

g 0-4 Cll .0 0 en ~ 0 -2

5 7 8

0 ~~0-----1~5-----3L0----4~5-----6L0----9L0--

Time, min

Fig. l-Effcct of brahma rasayana on lipid peroxidation in viiro -­(A) lipid pcroxidation induced by various process, and (B) time

course of lipid peroxidation.

c

;~ I

.g 0 o 2 4 6 B 10 12 :0

BO~ B :c ·~· E

~ 4~~- I I 0 I

0 50 100 150 200 250 30 0 3 50

60 f c .

~t~l I I 0 2 4 6 8 10 1 2 "4 16 18

Concentration of BR ( 1-1g/ml )

Fig.2-Effcct of brahma rasayana on oxygen radical generation in viiro -- (A) hydroxyl radical; (B) supcroxidc radical; and (C) nitric

oxide radical.

Table 2-- Effect of brahma rasayana(BR) n nitrite production and PMA induced superoxid.., generation in mice

peritoneal macrophagcs (in vivo ). [Values arc mean± SO of 3 observations]

Treatment Percent inhibition

BR (5x l0mg)

BR (5x50mg)

BR ( lx50mg)

Nitrite Superoxidc production

25.2 ± 2.5

37.8 ±9.6

ge-nerat ion

44.4 ±4.2

21.0±2.8

Percentage inhibi tion was calculated from control peri toneal macro phages on day 5.

REKHA et a/.: ANTIOXIDANT ACTIVITY OF BRAHM A RASA YANA 45 1

Concentration of BR needed for 50% inhibition was

found to be 5.5 J.lg/ml (Fig 2C) . Methanolic extract of BR was found to scavenge the nitric oxide radical produced by peritoneal macrophages. Concentration

needed for 50% inhibition was found to be 4J.lg/ml.

In vivo study

Inhibition of mtrrc oxide radical production in peritoneal macrophages by BR - In vivo treatment with BR was found to reduce the nitrite ions produced by macrophages(Table 2). Animals were treated with five doses of BR( lO and 50mg/dose/animal, po) was fo und to produce lesser nitrite ions; the percent inh ibition observed was found to be 25.2 and 37.8 respecti ve ly.

Inhibition of PMA induced superoxide generation in peritonealmacrophages by BR - BR was found to scavenge the superox ide generation Ill the macrophages(Table 2). Percent inhibit ion of superox ide generated by macrophages from animals treated with 5dail y doses of BR was 44.4% and from animals treated with one dose of BR inhibiti on was 2 1%.

Discussion

Even though herbal drugs and products derived fro m plants are still bei ng used in medical practice, the mechani sm o f action o f many herbal drugs are un known. Ac ti ve principle in these drugs are se ldom identified. In the case of po lyherbal preparations thi s problem is more acute as many drugs may synergisti ca ll y or antagonis ti ca ll y ac t together to g ive the fi nal ac tiVI ty of the preparat ion. He rbal preparations in Ind ian med ic ines(Ayurveda) have withstood the test of the time and are being practiced in Indi a along with the modern medicine . Herbal preparations in India are especia ll y useful aga inst of autoimmune di seases and many are i mmunorestorati ve.

Brahma rasayana(B R) is a polyherbal preparations with nearl y 60 plant ex tracts o f va rious concentrati ons bei ng used as a medicine to combat immunodefi ciency. A systemic admini stration of BR was fo und to improve the cell mediated humoral immunity in mice. Conventional therapy of cancer always produce side effec t and the most important being myelosuppress ion, which at times produce life threatening consequences. Brahma rasayana has been shown to protect the ti ssues from the undesirable side­effects of radiation4 and was found to reduce

myelosuppression Ill cancer patients undergoing chemotherapy 18

.

The present study indicate that BR could inhibit the oxygen radicals as seen from the inhibition of lipid peroxidation, scavenging of superoxide, hydroxyl radical and nitric oxide radical in vitro and in vivo. lt was also found to scavenge the lipid pe roxide already present in the ti ssue. BR was also found to scavenge both superoxide rad ical and nitrite ions (in vitro and in vivo ) produced in mice peritoneal macrophages . Present studies indicates that BR could reduce the oxygen radicals and subsequently reduce the harmful effects produced by the oxygen free radicals mediated Ill JUrieS.

References

I Sun Y, Free radicals, ant ioxidant enzymes and carcinogenesis. Free radical Bioi Med. 8( 1990)583.

2 Nish igaki I, Kuttan R, Oku H, Ashoori F, Abc H & Yagi K, Supprcsssivc effect of curcumin on lipid peroxidation induced in rats by carbon tetrachloride or Cobalt- 60 irradiation, 1 Clin Biochem Nwr, 13(1992)23.

3 Singh K, Concept of ojas and effect of rasayanas in the management of cancer, Proceeding of Ayurveda seminar on cancer, Thrissur IOtl' March, 15( 1990).

4 Pravccn Kumar V, Kuttan R & Kuttan G, Radioprotective effect of rasayanas, Indian 1 £rp Bioi, 34( 1996)848.

5 Pravccn Kumar V, KutLan R & Kuttan G, Chcmoprotcctivc action of rasayanas against cyclophosphamide toxicity, Tumori. 80(1994)306.

6 Pravccn Kumar V, Kuttan R & KutLan G, Effect of rasayanas, a herbal drug preparation on immune responses and its significance in cancer treatment, Indian 1 Exp Bioi, 37( 1999)27.

7 Pravcen Kumar V, Kuttan R & Kuttan G, Effect of rasayanas on cell mediated immune responses in tumor bearing mice, Indian 1 £rp Bioi, 37( 1999)23.

8 Menon L G, Kuttan R & Kuttan G, Inhibition of chemical induced carcinogenesis by rasayana, 1 £rp Clin Cancer Res, 15( 1996)24 1.

9 Pravccn Ku mar V, Kuttan R & Kuttan G, Effect of rasayanas in normal and tumor bearing mice, 1 Exp Clin Cancer Res. 13( 1994)67.

10 Ohkawa H, Ohishi N & Yagi K, Assay for lipid peroxides in ani mal tissues by thiobarbi turic acid reaction, Anal Biochem, 95( 1979)35 1.

I I Hal liwll B, How to characterize a biological antioxidant, Free Radic Res Conunw z. 9( 1990) I .

12 Me Cord J M & Fridovich I, Superoxidc dismutasc, an enzymatic function for erythrocuprcin, 1 Bioi Chem, 244( 1969)6049.

13 Green L C, Wagner D A, Glogowski J, Skipper P L, Wishnok J S & Tannenbaum S R, Analysis of nitrate, nitri te and (15

)

ni trate in biological flu ids, Anal Biochem, 126( 1982) 131 .

14 Marcocci L. Maguire J J, Droy-Lcfaix M T & Packer L The nitric oxide scavenging property Ginkgo bi loba extract EGb 761 ,

452 INDIAN J EXP BIOL.. MAY 2001

Biochem Biophys Res Commun, 201(1994a)748.

15 Ding A H, Nathan C F & Stuehr D J, Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages, J lmmwwl, 141 ( 1988)2407.

16 Brouet I & Ohshima H, Curcumin, an anti-tumor promotor and anti-inOammatory agent, inhibits induction of nitric oxide synthase in activated macrophages, Biochem Biophys Res Commun, 206(1995)533.

17 Dwi vedi P D, Verma A S & Ray P E, Induction of immune rejection of tumors by protein A in mice bearing

transplantable solid tissue Dalton's lymphoma tumors, lmmunopharmacol/nummotoxicol, 14(1992) I 05 .

18 Joseph C D, Praveen Kumar Y, Kuttan G & Kuttan R, Myeloprotective effect of a non- toxic indi genous preparation rasayana in cancer patients receiving chemotherapy and radiation therapy, a pilot study, J Exp Clin Cancer Res. 18(1999)3 .

19 Vaidya Jadavaji Trikanji Acharya, Ra ayana part( chapter I), Charakasamhita-Chikil sa sthanam (4'h edition), (Munshiram Manoharlal Publishers (P) Ltd, New Delhi), 1981,41.