Indian Journal of Experimental Biology Vol. 39, April 2001 , pp. 355-359

Effect of Maharishi Amrit Kalash an ayurvedic herbal mixture on lipid peroxidation and neuronal lipofuscin accumulation in ageing guinea pig brain

BPS Vohra*, S P Sharma & V K Kansal** & S K Gupta Laboratory of Nutritional Histopathology, Kurukshetra University, Kurukashetra, 136 119 India.

**Animal Biochemistry Division, National Dairy Research Institute, Kamal, India.

Received I April 1999; revised 3 January 2001

The effects of ayurvedic herbal mixture Maharishi Amrit Kalash(MAK) were studied on brain lipid peroxidation, oxy­gen consumption, and lipofuscin accumulation in 10 months and 32 months old guinea pigs. Brain regions studied were cerebral cortex, hypothalamus, cerebellum and spinal cord. Parameters assessed were lipid peroxidation, oxygen consump­tion , and lipofuscin accumulation. The endogenous lipid peroxide was found to be increased significantly (P<0.05) in the 32-month-old animals. Neuronal lipofuscin accumulation in the neurons of cerebral motor cortex, cerebellum and cervical spinal cord was increased (P< 0.05) in the older animals. Oxygen consumption was found to be decreased significantly(P< 0.05) in the 32-month old guinea pigs. Treatment with MAK at a dose of 500 mg/kg body weight daily for two months re­duced the lipid peroxidation and lipofuscin pigment accumulat ion significantly in brain regions and it also helped in restor­ing the normal oxygen consumption in the older animals. This indicates antioxidant properties of MAK.

The free radical attack on polyunsaturated fatty acids (PUF A) induces the irreversible and deleterious changes in the cell membrane, and this is believed to contribute to ageing'-3. The lipid peroxidation results in the formation of lipofuscin, which accumulates in cells with ageing4

.5

• The results on the lipid peroxidation have not been so consistent. Some authors have reported an increase in lipid peroxidation with age6

-9, while others have reported decline in lipid

peroxidation lO-13. Keeping in view such ambiguities

we applied two different approaches to measure the lipid peroxidation with age. First, endogenous lipid peroxidation measured in freshly prepared homogenate. Second the tissue homogenate is incubated in the presence of room air and then reacted with thiobarbituric acid. This second approach is felt to provide an assessment of potential substrate available for peroxidation. While, first approach indicates already formed lipid peroxides in the tissue l2

.

Maharishi Amrit Kalash(MAK) an herbal mixture' prepared according to the ancient ayurvedic formulation. MAK is available in two forms 'MAK-4 and MAK-5'.

MAK-4 is called ambrosia, it is in the form of tablets. MAK-5 is called nectar it is available in the

* Address for correspondence: Department of Neurology, School of Medicine, Box 295, University of Minnesota, 420, Delaware SI. SE. Minneapoli s MN 554 55. USA

form of paste. The different component of MAK-4 and MAK-5 are described in various publications I4

- ' 7 .

According to Charka Samhita(an ayurvedic medical text)19 there is no maximum dose of this formulation, it can be taken up to the amount till it does not disturb the normal food consumption. Many of such preparations in ayurveda are called Rasayanas. MAK has shown promises in protection against free radicals attack l5

-18 . Rasayanas are believed to strengthen the

body's resistance to infections and diseases and enhances longevity '9. The mode of action and the cellular effects of MAK are not precisely known. Therefore, in the present study the effects of MAK on lipid peroxidation, lipofuscin accumulation and the oxygen consumption by the animals were studied.

Materials and Methods Male guinea pigs (Dunkin Harley) of two age

groups 8 months and 30 months were used in the present study. Each group was subdivided in two sub groups, each consisting of 40 animals. One subgroup served as control and the other as the experimental group and was given a mixture of MAK-4 and MAK-5 in the ratio of I :20. The MAK mixture was given intragastrically with the help of a canula at a dosage of 500-mglkg-body weight daily at 11 .00 hrs for two months. Both the groups were fed pelleted food (Hindustan Lever Ltd., New Delhi) ad Libitum.

Thiobarbituric acid was purchased from Sigma chemical Co., USA. Other chemicals were purchased

356 INDIAN J EXP BIOL, APRIL 2001

from SRL or SO-fine chemical Co. Mumbai , India and were of analytical grade. The Ayurvedic preparation MAK was a generous gift from Maharishi Ayurveda Corporation Ltd., Faridabad, India.

Oxygen consumed by each animal from different animal groups was measured using an O2 consumption apparatus20.

After the drug administration, the animals were decapitated and the brains and the spinal cords were dissected immediately and rinsed in chilled normal saline. The following discrete regions : cerebral motor cortex, hypothalamus, cerebellum and brain stem (pons and medulla), cerebral hemisphere without cerebral cortex, hypothalamus and cervical spinal cord were used for measurement21 .

Homogenates were prepared in a ratio of Ig of wet tissue to 9 ml of 1.15% KCI by using a glass-potter Elvehjen homogeni ser. Thiobarbituric acid reactive substance was measured in 1000x supernatant of (I) freshly prepared tissue homogenate and (2) the tissue homogenate was incubated in air at 37°C for one hr. MOA, the end product of superoxide radical induced lipid peroxidation was quantitized by the thiobarbituric acid colored reaction22. Total soluble proteins were measured with method described by lowry et al 23

.

For histochemical studies, animals were perfused transcardiacally with EOTA (3% in physiological saline) for 10 min followed by formaldehyde- saline solution (10% in physiological saline) for 20 min as described by Zeman and Innes24

.

Motor cortices, cerebella and cervical spinal cords were dissected out and fixed in formaldehyde-calcium (FCA)25. For lipid histochemistry, animals were decapitated, various tissues were removed and quenched in liquid nitrogen . Latter 5-7I-lm thick sections were cut on Slee cryostat. The sections were stained in Sudan Black B (SBB), Periodic acid-schiff (PAS), ile blue sulphate (NBS), Schmorl and Ziehl

ee1son (ZN)26. This helped in studying the histochemistry of lipofuscin. Fluorescent microscopic studies were carried out with Olympus Fluorescent microscope equipped with UV light source (Osram HBO-200) and a filter combination of UG 1 exciter filter and L-420 barrier filter that give an excitation maxima at 366 nm and an emission maxima at 450 nm. The deparaffinised unstained and cryostat cut sections were studied. Sections were mounted in Olympus non- fluorescent immersion oil (nd 1.404 at 25°C) before observing under the microscope. The fluorescent pigment stood out sharply against the

cytoplasm. Using camera lucida attachment (under 40x objective and lOx eyepiece)neurons(numbering 100-300) along wit.h the fluorescing lipofuscin were drawn on a graph paper(having squares of I cm3 intersected with 10 divisions). The area of each neuron and that of lipofuscin were measured by a planimeter, having a zero-setting device, a polar compensation and an optical tracer system. Percentage cytoplasmic area occupied by the pigment was calculated for each neurons27 independently by four different persons. The data were finally taken and were statistically analysed for determining the significance. Student's 't' test was used to determine the statistical significance28.

Results Oxygen consumption was found to decrease

(P<0.05) in the 32 months old animals (Table I). The endogenous TARS as depicted in the Table 2 ranged from 26.78 nmole/mg protein to 76.89 nmole/mg

Table I-Effec t of MAK on oxygen consumpli on [Values, expressed as oxygen consumed ml/min/kg body wI. ±SE

of to animals)

Young animals (10 months)

Treated

Old animals (32 months)

Control Treated Control

86.46±1.34 89.81 ±t.20 49 .76±1.l 1 ** 64.43 *±1.73

*(P<0.05) compared wilh control with in the same age group. **(P<0.05) Compared with to months old control animal s.

Table 2-Effect of MAK on the level of endogenou TARS production

[Values, expressed as nmole of TARS/ mg protein, are mean±SE of 5 animals]

Tissue Young an i mals Old animals (10 months) (32 months)

Control Treated Control Treated

Cerebral 41.86 32.10* 82. 12** 42 .77*

cortex ±1 .27 ±t.1 8 ± 1.31 ±1 .O7

Hypothalamus 32.37 26.40* 87.93** 37.78*

±t.OO ±O.79 ±t.37 ±t.17

Rest of the 76.89 57.08* 122.55** 70.95*

Cerebrum ±1.39 ±t.ll ±t.43 ±t.57

Brain-stem 43.8 1 19.02* :12. 12** 24.47*

±t.07 ±O.78 ±t.59 ±t.71

Cerebellum 27.68 18.43* 50.57** 29.41 *

±t.76 ±O.99 ±1.0 1 ±O.78

Spinal cord 41.82 26.70* 81.40** 42. 17*

± 1.08 ±1.00 ± 1.l7 ±O.97

*(P<0.05) compared with controls within the same age group. **(P<0.05) Compared with 10 months old control animals

VOHRA et al. : EFFECT OF MAHARISHI AMRIT KALASH AN A YURVEDIC HERBAL MIXTURE 357

protein in different regions of eNS, the highest being in the rest of the cerebrum and the least in the cerebellum. The amount of endogenous TARS increased in old age (P<0.05) . The highest increase was observed in the hypothalamus, i.e. 76 .74% and the lowest in the rest of cerebrum, i.e. 59.38%.

The TARS production after incubat ing the tissue homogenate for one hour in air (in vitro) was lower in the tissues collected from the 32 months old animals (P<0.05) than in the 10 months old animals. The maximum difference was observed in spinal cord (60.88%) and brain stem (44.48%) and the minimum in hypothalamus (10.6%) (Table 3). The lipofuscin accumulation with age wa highest in cerebral motor

Table 3--Effect of MAK on the level of ill vitro TARS produc­tion

[Values. expressed as nmole of TARSI mg protein. are mean±SE of 5 animals]

Tissue

Cerebral cortex

Hypothalamus

Rest of the

Cerebrum

Brain-stem

Cerebellum

Spinal cord

Young animals ( 10 months)

Control Treated

15.85 10.37*

±D.17 ±D.33

12.52 08.21*

±D.27 ±D.31

20.20 16.07*

±0.23 ±I.IO

2 1.14 10.60*

±D.61 ±D.25

10. 17 07.71 *

±1.05 ±D. 77

15.39 06.02*

±D. IO ±D. 14

Old animals (32 months)

Control Treated

10.80** 05.12*

±D.53 ±0.24

10.26** 07.40*

±D.13 ±D. 18

14.98** 07.10*

±D.71 ±D.22

10.60* ±5.44*

±D.24 ±D.22

07.71** ±05.00*

±D.62 ±0.53

10.65** 05.80*

±D.18 ±O.21

*(P<0.05) compared with controls within the same age group. **(P<0.05) compared with 10 months old control an imals.

Table 4-Effect of MAK on the percentage neuronal are occupied by lipofusci n

[Val ues. expressed as percentage neuronal are occupied by lipo­fusc in ±SE of 10 animals]

Tissue

Cerebral

cortex

Cerebellum

Spinal cord

Young animal.. ( 10 months)

Control Treated

4.79 4.3 1

±0.32 ±D.36

4.2 1 4.05

±0.35 ±D.35

3.10 3.00

±D.35 ±D.34

Old animals (32 months)

Control Treated

7.3 1 ** 6.23*

±D. 50 ±D.41

6.89** 5.95*

±D.42 ±D.43

5.15** 4.31*

±D.32 ±D.34

*(P<0.05) compared with controls within the same age group. **(P<0.05) compared with 10 months old control animals.

Fig. I-Fluorescent micrograph from unstained motor cortex section of 32 months old guinea pig. Showing autofluorescent pigment aggregations (p) in pyramidal neurons at luxtanuclear posi tion. x 1520 Fig. 2--Fluorescent micrograph from unstained motor cortex section of 32 months old guinea pig treated with MAK Note very few fluorescent pigment granules. x 1050 Fig. 3--Photomicrograph from unstained cerebellum section of 32 months old guinea pig showing Purkinje neurons. Note heavy depositions of lipofuscin. x 1120 Fig. 4-Photomicrograph from cerebellum section of 32 months old guinea pig treated with MAK showing decreased amount of lipofuscin. x 1050 Fig. 5-Photomicrograph of cervical spi nal cord section showi ng anterior hom cells of 32months old guinea pig. Note heavy accu­mulation of lipofuscin x 320 Fig. 6--Fluoreseent micrograph of 32 months old treated guinea pigs showi ng anterior horn cells with decreased lipofu scin . x 320

358 INDIAN 1 EXP BIOL. APRIL 2001

cortex followed by cerebellum and spinal cord (Table 4 & Figs 1-6).

The treatment with MAK increased the oxygen consumption only in the 32 months old animals (P<0.05). The drug treatment decreased the level of TARS significantly in all the regions of CNS of both the age groups. The reduction was highest in the brain stem (70.20%) followed by the hypothalamus (57.02%). The reduction in the TARS was lower in 10 months old animals as compared to the 32 months old animals in each region of CNS. The drug treatment resulted in a reduction in the in vitro TARS production in both the age groups of animals (P<0.05).

The lipofuscin accumulation was found to be decreased to a significant level in the MAK treated groups (P< 0.05) (Figs.2, 4 & 6).

Discussion

The endogenous TARS was found to be increased with age in all the regions of CNS studied. The pres­ent study reveals lower in vitro TARS production in 32 months old animals. It appears that as the level of endogenous peroxides increases with age, the amount of substrate available for peroxidation, as measured by incubating tissue homogenate prior to reacting them with TBA, actually decreases with advancing age29

. This view is also supported by the observations that the ratio of unsaturated to saturated fatty acids decreases in membrane with age30-31. Thus, the en­dogenous lipid peroxidation is quite different from TARS produced by incubating the homogenates in the air. Treatment with MAK may be effective in pre­venting the free radical induced damage as it contains a large number of compounds namely, tannic acid, flavanoids, catecholamines, tocopherol , polyphenols, ascorbates, riboflavin, carotenes, mucilage, octacosa­nol , saponins, sphaeranthine, asparagine, glycyrrhizin, camphene, limonenc, pinene, etc. 13.l7

. Some of the above mentioned chemical components act as potent antiox idantsI5

.16. The lipofuscin accumulation was

found to decrease significantly in MAK- treated groups (P<0.05) . This may be due inhibition of lipid peroxidation because lipofuscin fo rmation results from lipid peroxidation . Hence it is concluded that MAK can be effective in checking the lipid peroxida­tion and decreasing the percentage neuronal area oc­cupied by lipofuscin. Since there are many other an ti­ageing compounds like dcprenyl and acetyl carnitine which has also shown their positive effects32

.36 MAK

increases the acti vity of various enzymes only in the

old animals in a very region specific wayl9. Oeprenyl increases the activity of superoxide dismutase (SOD) and catalase (CAT) but its effect on glutathione per­oxidase (GPx) is still a matter of controversi2

• Simi­larly acetyl carnitine also does not show any effect on the activity of GPX36. However the treatment with MAK increases the activity of SOD, CAT and GPX I9

•

Since GPx has lower Km value than CAT hence can be considered more effective in protection against H20 2

37. The reduction in the total oxygen consump­tion may have certain adverse effects on the Brain as the brain itself consumes 25% of the total oxygen consumed by the body38. Thus the normalization of oxygen consumption after MAK treatment in older animals may be helpful to normalize the physiological functions of the brain, although more studies are re­quired to establish this claim. Thus MAK appears to provide a good protection against free radicals like centrophenoxine deprenyl and acetyl carnitine37

•

References I Harman D. The free radical theory of age ing. Age.7 ( 1984)

Ill. 2 Cutler R. Free radicals and ageing. in Molecular basis of

ageing. edited by Roy and Chaterjee (Plenum Press. New York) 1984. 15.

3 Halliwell B. Free radicals. oxygen toxicity and ageing, in Age pigment. edited by RS.Sohal (ElsevierlNorth-Holland. New York)1981. 1.

4 Sharma S P. Gupta SK & Palro IK. Infl uences of centro­phenoxine on the anterior hom cells of protein malnourished wi star rats. Proc. NaIL Acad Sci India. 52(8)( 1987) 247.

5 Patro I K. Sharma S P &Patro N. Neuronal lipofuscin. its formation and reversibility. Indian Rev Ufe Sci. 8(1988) 95.

6 Noda Y. McGeer P L & McGeer E G. Lipid peroxides in brain duril/g ageing and vitamin £ deficiency: Possible rela­tiol/ to change in neurotransmitter indices. Ageing. 3( 1982) 173.

7 Mijuno Y & Ohta K. Regional distribution of thiobarbitu ric acid reactive products. activities of the enzymes regulating the metaboli sm of oxygen free radicals and some of the re­lated enzy mes in adult and aged rat bra ins. J Neurocllelll. 46(1986) 1344.

8 Gupta A. Hasan M. Cllander R & Kapoor N K. Age related elevation of lipid perox idation products: Diminution of su­peroxide di smutase activity in the central nervous system of rats. gerolltology. 37(6)( 1991) 305.

9 Rodriguez-Martinez M A. Alonso M 1. Redondo 1. Salaices M & Marin, 1. Role of li pid peroxidation and the glutathione dependent antioxidant system in the impairment of endothe­lium dependent relaxation with age. Br J Pllar/lla­col. 123( 1998) I 13.

10 Boehme D H. Kosecki R. Larson S. Stern F & Marks N. Lipid perox idation in human and rat brain ti ssue: Develop­mental and regional studies. Brain Res. 136( I 977) I I.

I I Devasagayam T P A. Senescence associated decrease of NADPH induced lipid peroxidation in r3t liver microsomes. F£BS Letters. 205(2)(1986) 246.

VOHRA et al. : EFFECT OF MAHARISHI AMRIT KALASH A A YURVEDIC HERBAL MIXTURE 359

12 Ansari A H, Kaplan E & Suhheman O. Age related changes in lipid peroxidation and protective enzymes in the centra l nervous system. Growth Dev & Age, 53( 1989) I 17.

13 Vohra BPS. Sharma S P, Kansal V K, lames T 1 & Reddy B N, High rate of lipid peroxidation in pons may be a possible cause of most of the pre-senile and senile brain disorders. In ­dian J Gerolllol. 7(3-4)( 1992) 51.

14 Oileepan K N. Patel V. Sharma H M & Stechschulte 01. Priming of splenic lymphocytes after ingestion of an herbal food supplement: Evidence for an immunomodulatory effect. Biochem Arch, 6(1990) 267.

15 Owivedi C, Sharma H M, Doborwoski S & Engineer F N, Inhibitory effects of Maharishi-4 and Mahari shi-5 on micro­somal lipid perox idation. Pharm Biochem Behav, 39( 1991) 649.

16 Sharma H M, Dwivedi C, Satler B C. Gudehithlu K P, Abou­Issa H, Malarkey W & Tejwani G A, Antineoplastic proper­ties of Maharishi -4 aga inst DMBA-induced mammary tumors in rats. Pharmacol Biochem Behav, 35(1990) 767.

17 Sharma H M, Lee 1 Y, Kauffman EM & Hanna A N, In vi tro effect of herbal mixture MAK-4 on antioxidant capaci ty of brain microsomes. Arch Biochem, 12( 1996)181.

18 Vohra BPS, Sharma S P, Kansal V K, Mahari shi Amrit Kalash rejuvenates antioxidant defence system in the ccntral nervous system of old guinea pigs. Pharmacol.Res, 40( 1999)497.

19 Charkasamhita of Agni vesh, rev ised by Carka & Ordhha­vala,19lh cd. by PI.Ka hinath shashtri 12-13. Varanasi. Ind ia: (Chukhmba Sanskrit Sansthan). 1993.

20 Rao K & Patnaik B, Correlation between body wcight. snout to vent length, tail girth and metabolic rate in male garden lizard, Calores versicolor. Exp. Gerontol. 8( 1973) 173.

21 Pearson R & Pearson L, The vertebrate brain (Academic Press, London) 1976.

22 Placer Z A, Cushman 1 & 10hnson B C, Estimation of prod­uct of lipid peroxidation, malondialdehyde in biochemical system. Anal Biochell1, 16( 1966) 359 ..

23 Lowry 0 H, Rosebrough N S, Farr A L & Randall , Protein measurement wi th phenol reagent. J Bioi Chem. 19J( 1951 ) 265.

24 Zeman W & Innes 1 RM, Craigie's neuroanatomy of rat(Academic Press, New York) 1963,pp 41.

25 Bancroft 1 0: An introduction to histochemical techniques. (Bullerworths, London) 1967.

26 Pearse AGE: Hisotchelllistry. theorerical and applied( Churchill London) 1972.

27 lames T 1 & Sharma S p, Geromology, 41 S2 (1995) 213. 28 Panse V G & Sukhatme P V. in Statistical /IIethods for (/gri ­

cullllral workers(lCAR Publications. ew Delh i) 1985. 29 Cand F & Verdetti 1, Superoxide dismutase, glutathione per­

oxidase, catalase and lipid peroxidation in the major organs of the ageing rats. Free Rad Bioi & Med, 7( 1989) 59.

30 Awad A B & Challopadhya 1 P. Developmental alterations in 5-nucleotidase kinetics and lipid composition of rat heart sar­colemma Mecll Age Dev, 24( 1983) 151.

31 Lewin M Band Timiars P S, Lipid changes in cardiac mito­chondrial membranes. Mech Ageing Dev, 24( 1984) 343.

32 Kitani K, Kanai S, Ivy GO & Carrillo MC, Pharmacological modifications of endogenous antioxidant enzymes with spe­cial reference to the effects o f deprenyl : A possible antioxi­dant strategy. Mech Ageing Dev.111 (2-3)( 1999)211.

33 Kitani K, Kanai S, Ivy GO & Carrillo MC, Assess ing the ef­fects of deprenyl on longevity and antioxidant defenses in different animal models. Ann N Y A cad Sci.20( 1998)854.

34 Gadaleta MN. Cormio A, Pe ce V, Lezza AM & Cantatore P. Ageing and mitochondria. Biochimie.80( I 0)( 1998)863.

35 Aureli T, Di Cocco ME, Capuani G, Ricciolini R, Manelli C. Miccheli A & Conti F, Effect of long- term feeding with ace­tyl-L-carnitine on the age-related changes in rat bruin lipid composi tion: a study by 31P NMR spectroscopy. Neurocilem Res.25(3)(2000)J95.

36 Kaur 1, Sharma 0 & Singh R. XVI annual conference of the Indian Academy of Neuro sciences(lAN) and National Sym­posium on Signal Transduction and Apoptosis in Developing and Ageing brain, Nov24-28,I998,24 AlIMS, New Delhi.

37 Bartsoz G & Bartkowiak A, Ageing of erythrocytes, II Ac­IIVltJes of peroxide detoxifying enzymes. Experien­tia,37(198 1)722.

38 Ordy 1 M& Kaack B, in Neurobiology of ageing: An inter­discipilillary life-spall approach, edited by K R Briz­zee(Plenum Press,New York and London) 1974.