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Indian Journal of Experimental Biology Vol. 39, December 2001 , pp. 1258-1262
Maharishi Amrit Kalash, an ayurvedic medicinal preparation, enhances cholinergic enzymes in aged guinea pig brain
BPS Vohra* & S P Sharma Laboratory of Nutritional Histopathology and Ageing, Department of Zoology, Kurukshetra Uni vers ity,
Kurukshetra 136 119.lndia
and
V K Kansal **
**Division of Biochemistry, National Dairy Research Institute, Kamal, . India
Received 25 May 2000; revised 3 September 200/
The effect of orally fed Maharishi Amrit Kalash was examined on the activities of cholinergic enzymes in the guinea pig brain. The acti vi ty of the cholinergic enzymes viz. choline-acetyl transferase and acetylcholinesterase enzymes was found to be reduced significantly (?<0.05) in the various regions of CNS of the aged guinea pigs. Oral administration of MAK(500 mg/kg body weight daily) for 2 months significantly increased (?<0.05) the activity of choline acetyltransferase and acetylcholinesterase in the older animals. The present study indicates that this food supplement can be helpful in alleviating the cholinergic deficits in the old age.
Maharishi Amrit Kalash is an Ayurvedic medicinal preparation. It is manufactured in two forms MAK-4 and MAK-5 . The main components of MAK are described elsewhere' -3. In brief it contains Terminalia chebula, Phyllanthus officianalis, Becopa monniera, Withania somnifera, Ipomea digitata, embelica officianalis, etc. The chemical analysis of MAK has shown that it contains a large number of compounds namely, tannic acid, flavinoids, catecholamines, atocopherol , polyphenols, ascorbates, riboflavin, Pcarotenes, mucillage, octacosanol, saponins, sphaer:mthine, asparagine, glycyrrhizin, camphene, limonene, pinene, etc '·3
. Some of the above-mentioned chemical components are potent antioxidants.
Previous experiments have shown that MAK reduces carcinoma in up to 88% of the experimental animals and causes regression of the 60% fully formed tumors 1, prevents lung cancer in metastasis in up to 65% of the animal's tested2
; enhances lymphoproliferative response in antigen-stimulated animals as compared to control ones3
; reduces platelet aggregation induced by adenosine diphorase, arachidonic acid, collagen, and epinephrine4; inhibits the opioid receptors and reduces the level of substance p. alleviates depression and stress5; inhibits the microsomal
*Correspondent address: Department of Neurology, Box 295, Uni versi ty of Minnesota, 420 Delaware St. SE, Minneapols, 55455, MN, USA
lipid peroxidation6·7
; decreases free radicals and reactive oxygen species, including superoxide, hydrogen peroxide, and hydroxyl radical , generated both in cellular (neutrophil) and non-cellular (xanthinexanthine oxidase systems8
; protects against the mitochondrial deterioration in the ageing brain9
; increases the mitochondrial9 and cytosolic antioxidant enzyme activities 10 and prevents neuronal lipofuscin accumulation' '.
A cholinergic deficit in the form of changes in the activities of the cholinergic neurotransmitter enzymes choline acetyltransferase and cholinesterase is often found in the ageing brain 12. Alterations in the cholinergic system are often considered a prominent feature of brain ageing 13
. These changes have often been found to underlie age-related impairment in cognitive(memory-learning) abilities. Cholinergic abnormalities have also been observed in alzheimer victims14"15 . Since MAK is believed to retard ageing, enhance memory and maximize longevity 16, it would be of interest to determine the MAK' s influence on cholinergic neurotransmitter enzymes in the ageing brain. The results will provide information of the mechanism by which MAK may exert its therapeutic/pharmacological effects.
Materials and Methods Male guinea pigs (Dunkin hartley) of two age
groups (8 and 30 months) were used in the present
VOHRA et al.: MAHARISHI AMRIT KALASH, AN A YURVEDIC PREPARATION 1259
study. Each group was subdivided in to two subgroups, of 40 animals each. One subgroup served as control and other was fed a mixture of MAK-4 and MAK-5 in the ratio of 1:20 in milk. The mixture was given with the help of a canula at a dose of 500-mg/kg-body weight daily at 11.00 hours for two months. The control animals received milk only. According to Charka Samhita 16 there is no maximum length of time for which this drug can be taken. Both the groups were fed pelleted guinea pig food (Hindustan Lever Ltd., New Delhi) and water ad libitum.
Animals (experimental and controls) were decapitated and the brains and the spinal cords were removed immediately and rinsed in chilled normal saline. The different regions of the brain, viz. the cerebral cortex, the hypothalamus, the cerebellum and the brain stem (pons and medulla) were separated. The cerebral hemisphere without the cerebral cortex and hypothalamus has been named as rest of the cerebrum in the text. Tissue samples were homogenized ( 10%) in 0.32 M sucrose solutions using a glass homogenizer. The homogenates were centrifuged at I 000 g for 6 min at 4°C. Supernatants were saved and a portion was used for assaying the acetylcholinesterase activity. Pellets were resuspended and homogenized again, and then centrifuged at 1500 g for 5 min. The supernatants obtained from both the centrifugation steps were combined and centrifuged at 10,000 g for 15 min 17
.
Acetylthiocholine iodide and acetyl CO A were purchased from Sigma Chemical Co., USA. Dithiobisnitrobenzoic acid was purchased from Fluka chemical Co. , Switzerland. Other chemicals were purchased from the CSIR center for biochemicals, New Delhi , SRL or SO-fine chemical Co. Mumbai , India and were of analytical grade. The Ayurvedic preparation MAK was a generous gift from the Mahari shi Ayurveda Corporation Ltd., Faridabad, India.
Choline acetyltransferase (EC 3.1.1.7) was assayed in IO,OOOg supernatant by a standard method 18 and the enzyme activity was expressed as 11mole of coenzyme A(CO.ASH) formed/min/mg protein. Acetylcholinesterase was assayed in 1,000 g supernatant according to the method of Ellman eta/. 19
• The enzyme activity was expressed as 11moles of acetylthiocholine hydrolyzed/min/mg protein. Protein was assayed by the method of Lowry et al. 20
. The results were statistically analyzed by Student's t test.
Results Results are presented in Tables 1 and 2. The activ
ity of both the enzymes ChAT and AchE, decreased
significantly with age in the brain regions studied. The decline in the activity of ChAT followed a negative rostrocaudal pattern as the maximum decline of the activity was in the cerebral cortex (63.21 %) and the lowest in the spinal cord (36.51%) followed by the cerebellum (25.39%). The treatment with MAK effectively increased the ChAT activity in all the regions of old animals. The maximum increase was in the cerebral cortex (88.19%) and hypothalamus (58.43% ). In the case of young animals, MAK effect was seen only in the brain stem. The activity of AchE also declined with age in the old animals and the minimum decline was observed in cerebellum (15.92%) followed by spinal cord (25.11%). The treatment with MAK increased the AChE activity in the brain regions of old animals only. The highest increase in the activity was found in the hypothalamus (61.18%). Interestingly MAK increased ChAT activity only in brain stem of the young animals, rest of the regions did not show any effect of MAK on either of the enzymes activities. We have used a nonradioactive assay method for choline acetyltransferase. Our results do not deviate much from the activity
Table !--Effect of MAK on the activity of choline acetyltransferase
[Values, expressed as !Jmole of CO.ASH formed/min/mg protein, are mean ±SE of 5 animals in each group ]
Tissue
Cerebral cortex
Hypo-thalamus
Rest of the Cerebrum
Brain-stem
Cerebellum
Spi nal cord
Young animals (10 months)
Control Treated
12.77 13.04 ±0.40 ±0.28
14.34 15.00 ±0.35 ± 1.10
12.32 12.58 ±0.41 ± 1.07
12.77 14.88* ±0.43 ±0.17
11.47 11 .83 ±0.21 ±0.21
11.38 12.36 ±0.33 ±0.31
Old animals (32 months)
Control Treated
4.69** 8.83* ±0.25 ±0.36
5.96** 9.88* ±0.30 ±0.30
7.16** 10.39* ±0.14 ±0.50
8.0 1 ** 11 .89* ±0.21 ±0.21
8.56** 10.47* ±0.33 ±0.42
7.22* * 10.46* ±0.16 ±0.41
*Values are significantly higher than those of controls within the same age group (P< 0.05) **Values arc significantly lower than those of young animal s (P< 0.05)
1260 INDIAN J EXP BIOL, DECEMBER 2001
Table 2-Effect of MAK on the activity of acetyl cholinesterase [Values, expressed as 11mole of acetyl thiocholine released/min/mg
protein, are mean ±SE of 5 ani mals in each group]
Tissue Young animals Old animals (10 months) (32 months)
Control Treated Control Treated
Cerebral 28.90 31.23 16.07** 24.08* cortex ±1.62 ±0.69 ±0.96 ±1.37
Hypo 33.52 35.22 12.9 1** 20.31* thalamus ±0.83 ±1.08 ±0.52 ±1 .08
Rest of the 30.04 31.55 15 .00** 21.29* cerebrum ±0.55 ±0.40 ±0.45 ±0.81
Brain-stem 25.90 27.65 19.42** 24.38* ±0.8 1 ±0.66 ±1.01 ±0.78
Cerebellum 26.05 29.48 21.90** 27.71 * ± 1.00 ±0.62 ±0.53 ±0.77
Spinal cord 5.22 27.79 19.63** 24.57* ±0.99 ±1.18 ±0.66 ±0.49
*Values are significantly higher than those of controls wi thin the same age group (P< 0.05) **Values are significantly lower than those of young animals (P< 0.05)
pattern observed by host of workers who have used radioactive methods21
-23
. The only difference which we have found is the activity of ChAT in the cerebellum. We have reported a decline in the activity of ChAT and AchE both with age in cerebellum of guinea pigs but the others have reported no change in the cholinergic enzyme activities with age in cerebellum of rat and mice24
'25
, this difference may not be due to the different techniques but may be due to different animal model than ours. Thi s can further be explained by the studies on fi sh brain, in which ChAT activity declined in all the regions26
•
Discussion Data obtained in the present study showed that
ageing decreased the activity of cholinergic neurotransmitter enzymes in the guinea pig brain . This is in agreement with studies on other animals21
-23
• Dysfunction of cholinergic system plays a major role in memory loss in aged individual and many workers have established a positive correlation between the loss of cholinergic enzyme activities and decrement in learning and memory. The loss of cholinergic neurons is well documented in Alzheimers's disease and other
behavioral deficits 12-
15'21
'23 that are more directly re
sponsible for cognitive impairments27• Thus the pres
ent findings are in agreement with the reports that a decrease in the cholinergic enzymes in CNS is a significant event in aging 13
-15
.
An increase in free radical attack during ageing may be responsible for the dysfunction of the cholinergic neurotransmitter enzyme system. Increase in membrane peroxidation adversely affects the · release of acetylcholine in rats28
• Lipid peroxidation decreases membrane fluidity which, in turn, reduces choline uptake by 60-70% in the neurons of aged rats29
. The transport of choline across the blood-brain barrier is also decreased in aged rats30
. Thus, both the diminished availability of the precursor and the decreased activity of free choline acetyltransferase may be responsible for the diminished neurotransmitter synthesis in the ageing brain31
• Antioxidant treatments enhance the activity of cholinergic enzymes12
·31
. We have earlier reported higher lipid peroxidation product accumulation in brain regions of guinea pigs9
-11
, and MAK because of its antioxidant properties lowered the risk of lipid peroxidation in the brain of guinea pigs. Therefore, the enhanced activity of ChAT and AchE in the aged guinea pigs after MAK treatment may be attributed to the antioxidant action of MAK.
Many other herbal and synthetic formulations have been reported to be effective in enhancing the cholinergic enzyme activities in aged animals. For example Kami-Untan-To(KUT), a Japanese herbal drug increases the ChAT activity in aged mice in cortex, striatum and hippocampus only32
, but it was not effective in Cerebellum and spinal cord. Nimodipine enhanced the activities of both ChAT and AchE in the brain of 3 months as well as 11 months old mice33
,
whereas MAK was very specific for its action on the aged animals only. Centrophenoxine another synthetic drug has been found to increase the AchE activity in the brain stem but not in cerebellum of the aged rats25
.
Similarly N(5-hydroxynicotinoil)glutamic acid(ONK) also increases the cholinergic enzyme activity in both the young and older animals34
• Acetyl-L-carnitine treatment also increased the ChAT activity in the aged rat brains35
. The mechanism of action of MAK seems to be similar to the action of another herbal formulation Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960), which has scavenging action against hydroxyl radicals, superoxide radicals and carbon centered radicals, decreases thiobarbituric acid reactive substance and increases SOD and cholineacetyl- transferase activities in the hypothalamus and hippocampus
YOHRA et al.: MAHARISHI AMRIT KALASH, AN AYURVEDIC PREPARATION 1261
of aged rats36. Similar effects for MAK on various
antioxidants properties have been observed9-
11• MAK
appears unique in its action as it increases the cholinergic activity in all the regions of brain tested in the older animals where cholinergic activity was significantly reduced. This appears to be a special property of MAK that it was effective only in the aged regions where normal homeostasis of the body is disturbed.
References I Dileepan K N, Patel V, Sharma H M & Stechschulte D J.
Priming of splenic lymphocytes after ingestion of an herbal food supplement : Evidence for an immunomodulatory effect. Biochem. Arch, 6 (1990) 267.
2 Dwivedi C, Sharma H M, Doborwoski S & Engineer F N. Inhibitory effects of Mahari shi-4 and Mahari shi-S on microsomal lipid perox idation. Phannacol Biochem Behav, 39 (199 1) 649.
3 Sharma H M, Dwivedi C, Satter B C, Gudehithlu K P, Aboulssa H, Malarkey W & Tejwani G A. Antineoplastic properties of Mahari shi-4 against DMBA-induced mammary tumors in rats. Pharmacal Biochem Behav, 35 (1990) 767.
4 Patel V K, Shen N, Sharma H M & Brahmi Z. Reduction of metastas is of lewis lung carcinoma by an ayurvedic food supplement in mice. Nut Res, 12 ( 1992) 5 1.
5 Sharma H M, Feng Y & Panganamala R Y. Mahari shi Amrit Kalash prevents human platelet aggregation. Clin Ter Cardiovas. 8 (1989) 227.
6 Hanissian S K,. Sharma H M & Tezwani G A. Effect of Mahari shi Amri t Kalash (MAK) on brain opioid receptors. FASEB J, 2 ( 1988) 433.
7 Sharma H M, Lee J Y, Kauffman E M & Hanna AN. In vitro effect of herbal mi xture MAK-4 on antiox idant capacity of brain microsomes. Biochem Arch, 12 ( 1996) 18 1.
8 Niwa Y, Effect of Maharishi 4 and mahari shi 5 on infl ammatory medi ators with special reference to their free radical scavenging effects. Ind. J Clin Pract. I ( 199 1) 23.
9 Yoh ra B P S, Sharma S P & Kansal V K. Effect of Mahari shi Amrit Kalash on age dependent vari ations in the mitochondrial anti ox idant enzy mes, lipid perox idation and mitochondria l popu lation in the di ffe rent regions of Central Nervous System of Guinea pig. Drug Metabl Dmg Inter, 18 (200 I) 57.
I 0 Yohra B P S. Sharma S P & Kansal V K. Maharishi Amri t Ka lash rej uvenates ageing central nervous system's anti ox idant defence system: An in vivo study. Phamwco/ Res, 40 ( 1999) 497.
I I Yohra B P S, Sharma S P. Kansal V K & Gupta S K. Effect of Mahari shi Amrit Kalash an ayurvedic herbal mixture on lipid perox idation and neuronal li pofuscin accumula tion in agei ng guinea pig brain. Indian J Exp Bioi, 39 (200 I) 355.
12 Maneesub Y, Sanvarinda Y & Govitripong P. Part ial restorati on of choline acetyltransferase act ivities in aging and AF64A- lesioned rat brains by vitami n E. Neurochem In/ , 22 (1993) 487.
13 Squire L R & Davis H P. The pharmacology of memory: a neurobi olog ical perspecti ve. Annu. Rev Pharmaco Toxicol. 2 I ( 1981) 32J.
14 Wilcock G K. Esiri M M. Bowen D M & Smi th C CT. Alzheimer's di sease. Correlat ion of cortical choline acety ltrans-
ferase activity with the severity of dementia and histological abnormalities. J Neural Sci, 57 (1982) 407.
15 Soncrant T T, Holloway H W, Greig N H & Rapport S I. Regional brain metabolic responsivity to the muscarinic cholinergic agonist arecoline is similar in young and aged Fischer-344 rats. Brain Res, 487 (1989) 289.
16 Charka Samhita of Agnivesh, rev ised by Carka & Drdhhavala, 19'h ed. by Pt. Kashinath Shastri . (Yaranasi, India).
17 Li H L, Moreno-Sanchez R, & Rottenberg H. Alcohol inh ibits the acti vation of NAD-Iinked dehydrogenases by calcium in brain and heart mitochondri a. Biochim Biophys Acta. 1236 (1995) 306.
18 Chao L P. Spectrophotometric determination of choline acetyltransferase in the presence of dithiothreitol. Anal Biochem. 85 ( 1978) 20.
19 Ellman G I, Courtney K D, Andres V Jr & Featherstone R M. A new rapid colorimetric determination of acetylcholi nesterse activity. Biochem Phannacol, 7 ( 196 1) 88 1.
20 Lowry 0 H, Rosebrough N S, Farr A L & Randall R. Protein measurement with phenol reagent. J Bioi Chem, 193 ( 195 1) 265.
21 Dravid A R. Deficits in cholinergic enzymes and muscarinic receptors in the hippocampus and striatum of senescent rats: Effect of chronic hydergine treatment. Arch lnt Pharmacodynm, 264 ( 1983) 195.
22 Michalek H, Fortuna S & Pinter A. Age-related di ffe rences in brain choline acetyltransferase, cholinesterases and muscarinic receptor sites in two strains of rats. Neurobiol Aging, 10 (1989) 143.
23 Wortwein G, Yu Juan, Toli ver-Kinsky T & Perez-Polo J R. Responses of young and aged rat CNS to parti al cholinergic immunolesions and NGF treatment. J Neurosci Res, 52 ( 1998) 322.
24 Vijyan V K. Cholinergic enzy mes in cerebellum and hippocampus of senescent mou5e. Exp Gerontal, 12 ( 1977) 7.
25 Sharma D & Singh R. Centrophenox ine ac ti va tes acetylcholinesterase ac ti vity in hippocampus of aged rats. Indian J Exp Bioi. 33 ( 1995) 365.
26 Bellanger C, Dauphin F, Belzunces L P, Cancian C & Chichcry R. Centra l acetylcholine synthesis and cataboli sm acti viti es in the cuttlefish during aging. Brain Res, 762 ( 1997) 219.
27 Shih Y H & Pugsley T A. The effects of various cogni tionenhancing drugs on in vitro rat hippocampal synaptosomal sodium dependent high affinit y choli ne uptake. Life Sci. 36 ( 1985)2 145.
28 Meyer E M & Judki ns J H. Effects of membrane peroxidation on [3H]acetylcholine release in rat cerebral cort ical synaptosomes. Neu rochem Res, 18 (1993) 1047.
29 Ghosh C, Dick R M & Ali S F. Iron/ascorbate- induced lipid perox idat ion changes membrane flu id ity and mu~carin i c
cholinergic receptor binding in rat fron tal cortex. Neurochem In !, 23 (1993) 479.
30 Hi cks M & Gebick i J M. Inhi bi tion of peroxidat ion in linoleic ac id membranes by nit rox ide radicals, butylated hydroxytolur:ne, and alpha-tocopherol. Biochem. Biophycs. Res Commw l. , 80 ( 1978) 704.
31 Giacobini E. A new hypothesis of the aging of cholinergic sy napses. in Cell ular and molecular mechanism of aging in the nervous system, ed ited by Giacobini , E. , Filogamo. G. and Vernadkis, A.( Raven Press, New York ) l982.
32 Wang Q, Iwasaki K, Suzuki T, Arai H, Ikarashi Y. Yabe T. Toriizuka K, l lanawa T, Yamada II & S:.s . .iki H. Potent iation of brain acetylcholine neurons by Kami-Untan-To (KUT) in
1262 INDIAN J EXP BIOL, DECEMBER 2001
aged mice: implications for a possible antidementia drug. Phytamedicine, 7 (2000) 253.
33 Kabuto H, Yokoi I, Mori A, Murakami M & Sawada S. Neurochemical changes related to ageing in the senescenceaccelerated mouse brain and the effect of chronic administration of nimodipine. Mech Age Dev, 80 (1995) l.
34 Taglialatela G, Navarra D, Cruciani R, Ramacci M T, Alema G S & Angelucci L. Spatial memory and NGF levels in aged rats: Natural variability and effects of acetyi-L-carnitine treatment Exp Gerontal, 29 (1994) 55.
35 Voronina T A, Garibova T L, Trofimov S S, Sopyev Z A, Petkov V D & Lazarova M B. Comparative studies on the influence of ONK (N(5-hydroxynicotinoil) glutamic acid), piracetam and meclofenoxate on the learning- and memoryimpairing effect of scopolamine, clonidine, and methergoline. Acta Physial Pharmacal Bulg, 17 (l99 1) 8.
36 Hiramatsu M, Edamatsu R & Mori A. Free radicals, lipid peroxidation, SOD activity, neurotransmitters and choline acetyltransferase activity in the aged rat brain. EXS, 62 (1992) 213.