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.

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