Screening Indigenous Medicinal Plants of Northeast India for Their Anti-Alzheimer ’ s Properties

Introduction: Alzheimer’s disease (AD) is a progressive neurologic disease of the brain that affects intellectual abilities, reasoning and memory. Acetylcholine (ACh) is involved in the maintenance of cognitive process. Pathologically, ACh production is compromised in the brains of AD affected people. Presence of acetylcholinesterase (AChE) in the synaptic cleft, which hydrolyzes ACh, further decreases the ACh-levels, and thereby, additionally compromises cognition. The tribal people of North East India have been using indigenous plants as traditional medicine for brain disorders. We assayed whether the plants used in the traditional tribal knowledge for the treatment of brain disorders might contain better AChE-inhibitors. Methods: We collected 10 traditional medicinal plants from Northeast India. A total of 39 plant extracts were prepared using three solvent systems. The Acetylcholinesterase (AChE) activity was measured with Ellman method. The experiment was done in triplicate for each level of inhibitor. The activity was measured at 412 nm wavelength using Plate Reader. The standard student t-test was used to show significant difference in IC50 values between extracts. Results: The result are reported based on Km, Vmax, IC50 (μg/μl), percentage inhibition and inhibition pattern. Two extracts had competitive inhibition, 11 extracts had mixed inhibition, 2 extracts had non-competitive inhibition, 11 extracts had uncompetitive inhibition and 4 extracts did not provide any proper pattern. The IC50 for these plant extracts were at the range of 0.51-12.4 μg/μl. Notably, Cinnamomum camphora (leaf: chloroform), Litsea glutinosa (stem; chloroform), and Litsea glutinosa (stem; methanol) showed IC50 values of 0.51, 0.53 & 0.81 μg/μl, respectively.

and accepted as the foremost hypothesis in the field of AD. 911 However, relationships between levels of acetylcholine and AD have been a challenge.It is now accepted that the acetylcholine dysfunction may not be the primary cause of AD but may be a consequence of the disease. 9he neurotransmitter acetylcholine is involved in active maintenance of novel information and enhancement of longterm potentiation, i. e. memory. 12he action of acetylcholine is terminated in the synaptic cleft by AChE which hydrolyzes ACh into acetate and choline.Amyloid plaques formation in synapse prevents acetylcholine molecules to reach its cognate receptors on the postsynaptic neuronal membrane, thereby, leading to the gradual loss of communication between neurons.Over the years several studies have focused on "cho linergic hypothesis" to ameliorate the acetylcholine deficiency in the brain of AD patients.These endeavors have led the scientists to discover various classes of molecules, for the treatment of AD, in the form of acetylcholine esterase inhibitors. 9Drugs based on cholinergic hypothesis work in prolong ing the duration of acetylcholine in the synaptic cleft by inhibit ing AChE activity.Some of these drugs have been synthesized and/ or derived from plants such as rivastigmine, huperzine and galan thamine etc. 1315 However, these drugs only slow down the progression of the disease and have been reported for various side effects such as gastrointestinal disturbances and bioavailability problems. 16,17These disadvantages make a room for finding newer drugs with better efficacy and lesser side effects.In traditional practices of Ayurvedic medicine, numerous plants have been used and studied as treatment for cognitive disorders including neurodegenerative diseases such as AD, memory enhancement, anti aging and preventing dementia. 18For example, Celastrus paniculatus Wild.(CP) has been reported to be used in Ayurvedic medicine for stimulating intellect and sharpening the memory. 18Centella asiatica (L.) Urb.(CA) is being used for antiaging, prevention of dementia and mental exhaustion. 18,19Acorus calamus L. (AC) extract is applied for the treatment of memory loss. 18,19whiles the ripe fruit of Terminalia chebula Retz.(TC) is regarded to slow down the ageing process and to improve the cognition. 18,19Recently Mucuna pruriens (L) DC has been shown to have potential for the treatment of Parkinson's disease 20 and Withania somnifera (L.) Dunal (WS) is routinely used for improvement of memory and cognition enhancement in Indian Ayurvedic medicine. 21The indig enous tribal people of North East India have been using various plants as traditional medicine for treating brain & neurological disorders, over the millennium.Some of these plants have been shown to have AChE inhibition activity. 22In addition, Semecarpus anacardium L. f. (SA) (from NorthEast India), has been shown to be neuroprotective especially to the hippocampal region in stressinduced neurodegeneration. 23iven that many current AD drugs are derived from plants and the North East India belongs to one of the major biodiversity hotspots region in the world, it would be justifiable to explore whether the plants used in the traditional tribal knowledge for the treatment of various brain ailments might contain better AChEinhibitors.To this end, we have collected several such plants and assayed their extracts for its capacity to inhibit AChE activity.The AChEinhibition activities of these plant parts were compared with the standard AChEinhibitor drugs and demonstrate that some of these plants contain a good amount of AChE inhibition activity that may be further investigated for isolation of activeinhibiting compo nent in the AD drug discovery process.

Plant material Collection & extraction
Under the aim of this study, different botanical books and journals were used to find the plants that are used either to treat any kind of brain disorder and/or improve the functions of brain by traditional healers in NorthEast India.A database of such plants was created that includes habitat, botanical and local name, purpose of use, picture of the plants and the journal or books where it was documented.Ten plants were collected from the gardens of Botanical Survey of India (BSI) which are located in the countryside of Shillong city in Meghalaya, India (with permission of Dr. B. K. Sinha, BSI Director on 03.10.2012).Proper identi fication and authentication of the collected plants were carried out with the help of specialist from BSI, and respective vouchers were provided.It is worth mentioning that although the plants which were collected from BSI and adjoining area of Shillong these plants may be found in other parts of India but we selected because these plants are being used by traditional healers of North East India for brain disorders.The plants were first washed to remove dust and insects and then trans ferred to the clean room (out of sunlight) to dry gradually.When the plants were completely dried, different parts of the plants including, leaves, stem, root, bark, or the whole plant were pulverized and trans ferred to autoclavable bottle with some silica gel to protect them from moisture.The bottles were labeled by the name of the plants and its part.
The plant parts were extracted using three solvents including water, methanol and chloroform.The plant powder/solvent was used at the ratio of 1 g/20 ml.The plant powder was mixed with the solvent and the bottle was sealed completely.The mixture was kept in water bath overnight at the temperature of 318 K, 318 K and 338 K for methanol, chloroform and water extract, respectively.The mixture was filtered using Whatman filter 40 (110 mm) gently.The solvents were removed using the evapora tor.In order to keep the same conditions and use the same amount of extracts in experiment, all extracts were lyophilized to remove moisture and get the extract completely dry.All extracts were sealed and kept at 4˚C.

Buffer and reagents preparation
The phosphate buffer 0.1M was prepared at pH 7 and pH 8. Acetylcholine esterase enzyme and Acetylthiocholine (ATC) substrate were purchased from Sigma.The lyophilized AChE was dissolved in 1% gelatin to obtain 1000U stock enzyme.The final enzyme concentration in the assay buffer was 1.4 U. Eight different final concentrations of ATC were adjusted ranging between 35.476 µM and 354.770 µM.The stock was prepared in distilled water in buffer pH 8.The DTNB (5, 5'dithiobis(2nitrobenzoic acid)) was obtained from SRL, India and used as a reporter reagent.The reaction between DTNB and free thiol group produces a mixed disulfide with thiols, liberating the chromospheres 2nitro5 thiobenzoate anion (TNB 2 ) which gives an intense yellow color at 412 nm with a higher molar extinction coefficient.The DTNB was dissolved in 0.1M phosphate buffer pH 7 in which it is more stable and used at a final concentration of 0.33 mM.
The stock solution of each plant extract, 5 mg/ml, was prepared by dis solving the powder in the phosphate buffer pH 8. Extracts were dissolved by pipetting and vortexing gently, following a short spin to remove the particles.The supernatant was used as experimental solution.The extracts were used at final concentrations of 0.8298 µg/µl and 1.0373 µg/µl.However, we would like mention that, in most of the cases, extracts dissolved in the phosphate buffer to a limited extent ranging from 30 to 100%, and hence the final concentrations used (0.8298 µg/µl and 1.0373 µg/µl) might be much lower.Tacrine, a first generation FDA approved AD drug and is a known noncompetitive AChE inhibitor 24,25 was used as a standard AChEinhibitor control.

Experimental Procedure
The Acetylcholinesterase (AChE) activity was measured with the method developed by Ellman et al., 1961 and modified by Laura et al., 2010.The experiment was set up for the final volume of 120.5 µl using 96well plate (Greiner bioone).The activity was measured at the wavelength of 412 nm using Plate Reader (Synergy H1 422, BioTek).The raw data was analyzed using OriginPro8 and Microsoft Excel software.

Initial Velocity (ν 0 )
The initial velocity (V 0 ) was calculated based on formation of the product at the particular time in the system.The rate or (V 0) is: = V0 = ∆[P]/∆t where [P] is concentration of product at time t 27 .According to the Beer Lambert Law [P] = A/εl, where A is absorbance, ε is molar extinction coefficient and l is light path length.The original molar extinction coeffi cient of DTNB which was reported by Ellman is 13,600M 1 cm 1 at 412 nm.However, later studies have shown that the more accurate molar extinc tion coefficient value is 14,150 M 1 cm 1 at 412 nm, which has been used in this study. 28,29And l for 120.5 µl (Greiner bioone plate) is 0.3374 cm.]/IC 50 % Inhibition 0.    V max and K m of control assay were 6.1 and 120 respectively (8 substrate concentrations).V max and K m of control assay were 6.5 and 139 for those which have been marked with star (*) (7 substrate concentrations).K i has not been mentioned here as it was not useful to compare the inhibition activity between two different extracts.Tacrine has been reported based on nanomolar.Water: ( w ), Methanol: ( m ) and Chloroform: ( c ).

Plots
The MichaelisMenten plot was graphed using V 0 versus [S].The Line weaverBurk plot was used to calculate V max (maximum velocity) and K m (Michaelis constant).The initial velocity of AChE was calculated for each substrate concentration individually.V max (No inhibitor), app max V (with Inhibitor), K m (No inhibitor) and app m K (with Inhibitor) were calculated by plotting the graph of 1/V 0 versus 1/[s].The proper inhibition pattern for each level of inhibitor was plotted.K i (Inhibition constant), %I (%Inhibition) and IC 50 was calculated accordingly.
To calculate the %I the following equation was used: Where (V i / V 0 ) is the fractional activity therefore [1 -(V i / V 0 )] is the fraction of enzyme occupied by inhibitor.V i and V 0 are reaction velocity at the presence and absence of inhibitor. 27e following equations were used to calculate IC 50 and relationship between [I]/ IC 50 and %I of enzyme activity: 27 Where [I] is the concentration of inhibitor (extract) and h is Hill coeffi cient which is related to number of active site of the enzyme and interac tion between inhibitor and enzyme.
After rearranging the equation E2 the following equation can be used to calculate IC 50 : Where, (V 0 / V i ) is reciprocal of fraction activity.
Looking at the equation E4 we see that at the point of 50% inhibition, the fractional activity will be 0.50 and its reciprocal will be equal to 2.00.It means when 50% inhibition is achieved then [I] / IC 50 is equal to 1.00.And if the [I] / IC 50 is calculated for different %I (i.e.25%, 30%, 50%, 75% and etc.), given h is equal to 1, the standard values as mentioned in Table 1 can be obtained. 27 rearrangement of the standard Langmuir isotherm equation the rela tionship between [I] / IC 50 and %I for enzyme inhibition was calculated: 27 [I] / IC 50 = %I / (100 %I) (E5) The standard values mentioned in Table 1 were used to evaluate whether our calculation is correct 27 (Table 1).
In order to show that how large a difference in IC 50 between two extracts can be considered significant, the standard student ttest was used: 27,30  Where, a and b identify two inhibitors (extracts), and S a and S b are stan dard errors of each IC 50 value, n a and n b are number of data points for two inhibitors (extracts) respectively.As our experiment was set up in triplicate, therefore, the degrees of free dom for testing the significance of tvalue was calculated using following equation: 27,30 df = {[(n a *3 Where n a and n b are number of data points for two inhibitors (extracts) respectively.
The experiment was set up for three levels of inhibitors (extract) 0.0000, 0.8298 and 1.0373 µg/µl.The activity without any inhibitor was nega tive control and Tacrine as known inhibitor was used as positive control.
The experiment was done in triplicate for each level of inhibitor plus one column (eight wells) as reference (R) without enzyme.Because of non enzymatic reaction between DTNB and substrate or inhibitors some absorbance will be detected as background which must be subtracted from the activity columns.Therefore, the reference column must include all reagents present in activity columns except enzyme (Figure 1).

RESULTS
A total of 30 extracts of 9 plants were screened for AChE inhibition activity.The inhibition pattern was found for each extract by plotting 1/V 0 versus  2).
Working with natural plant inhibitors is very cumbersome as different molecules are present in plant extract.Hence, using different concentra tion of the same crude extract might show variant inhibition patterns.Therefore, it is often difficult to find an inhibition pattern that may be appropriate for a certain plant extract and unlike pure compounds it is impossible to use K i to compare the inhibition activity of the plant extracts.In fact, even in pure compounds if they do not have similar inhibition pattern, K i cannot be a proper parameter to compare.Based on the type of reaction between inhibitor and enzyme, there are different types of inhibition patterns and different types of calculations for K i .
Having said that, it seems it won't be correct if we use K i as a comparison parameter to select the inhibitor.That is why we have not mentioned K i values in the Table 2, although the values have been calculated for all extracts.Therefore, it is necessary to have a particular comparison parameter which should have two important aspects.First, it should be indepen dent parameter that is it should not be influenced by the type of reaction between inhibitor and enzyme.Secondly, it should be applicable for both pure and crude samples.Practically in inhibition kinetics, it seems per centage inhibition (%I) and IC 50 /EC 50 are the best parameters which can be used to select inhibitors.The %I and IC 50 /EC 50 are using velocities directly (as first step values) and they are independent from the type of reaction between inhibitor and enzyme while K m and V max (as second step values) are necessary to calculate K i and they are not independent.When we work with crude samples, we have to consider the following things.Firstly, we assayed the crude plant extract we do not have any idea of what would be the real percentage of the active compound (that inhibits AChE) in the crude extract.Generally, percentage of any active compound in plant extract is much less than 1%.Secondly, because the crude contains several other compounds, these compounds will effectively inhibit and/or dampen the access of the active compound for AChE site and thus may display lesser inhibition capacity.Third, the active com pound might be in complex with other compounds which will prevent the reaction between active compound and AChE and reduce the inhi bition activity.Fourth, being in complex condition might change the solubility of active compound leading to reduction of active compound concentration in the stock solution.Fifth, as we are using crude plant extracts there might be some compounds which can increase the AChE activity, thereby reducing inhibitory potential of the activeinhibiting compound.Therefore, keeping all these in mind, it is not fair to compare the inhibitionactivity of a pure compound that with crude extract.In this study the best IC 50 with 0.51 µg/µl belongs to Cinnamomum camphora (leaf: chloroform) following by Litsea glutinosa 0.53 µg/µl (stem; chloroform), Litsea glutinosa 0.81 µg/µl (stem; methanol) and Litsea glutinosa 1.31 µg/µl (leaf; Water) (Table 2).We found 20 plant extracts having more than 20% inhibition and specifically the above mentioned plants displayed % inhibition range varying from 4054%.Other studies on different plants have reported more than 70% inhibition using plant extract concentration of 1 mg/ml. 16,38But first, none of them clarified for how they have done the analysis and calculations for %I and IC 50 using the velocities and inhibitor concentrations and what was the relationship between all these parameters.Secondly, %I is not a good parameter to compare two different plant extracts because it is almost impossible to use the same amount of different plant extracts due to 11 extracts had uncompetitive inhibition and 4 extracts did not provide any proper pattern (Table 2).AChE has Michaelis constant of 90 µM. 31 In this study, the maximum velocity and Michaelis constant for control assay (zeroinhibitor) were obtained as 6.1 and 120 µM, respectively, using eight different substrate concentrations.However, only for four assays we used seven different substrate concentrations and their V max and K m were of 6.5 and 139 µM.Based on obtained initial velocities and substrate concentrations, six different parameters including %I, IC 50 , [I]/ IC 50 , Ki, V max and K m were calculated for each extract.V max and K m were used to prove that the inhibition pattern has obtained correctly.Three parameters including %I, IC 50 and [I]/IC 50 were used to compare the inhi bition activity of extracts.We calculated a very important parameter ([I]/IC 50 ) and its relationship with %I.We compared our results for ([I]/IC 50 ) and %I with the standard values mentioned in Table 2.We found that relationship values between ([I]/IC 50 ) and %I of our data was in good agreement with the standard values.This indicates that our calculation for %I and IC 50 are correct.In order to show that how much difference between two values of IC 50 is considered significant, the Student ttest was done for those which have shown more than 20% inhibition.The t values were calculated at α = 0.05 and α = 0.01 (2 tail) (Table 3). 27,30 e Lineweaver-Burk and MichaelisMenten plots have been shown in Figures 2, 3 and 4 for mixed inhibition, uncompetitive inhibition, com petitive inhibition and noncompetitive inhibition.The plots for Tacrine, as a known AChE inhibitor, have been shown in Figure 5 to demonstrate that the enzyme assay was working correctly.

DISCUSSION
Pathologically, the formation of Amyloid plaques in synapse impedes the access of acetylcholine molecules to reach their cognate receptors on the postsynaptic membrane to deliver their message.In fact, gradual build up of Amyloid plaques in the synapse slows down the action of acetylcholine and leading to the loss of communication between neurons.Inhibiting AChE would mean that more acetylcholine molecules would be available in the synaptic cleft to deliver the message before they are being catalyzed by AChE.Therefore, to increase the acetylcholine levels in the brains of AD patients, search for AChEinhibitors has led to the discovery of plant derived drugs such as Rivastigmine, huperzine and Galanthamine.Rivastigmine and Galanthamine have been isolated from two different plants, Calabar bean (Physostigma venenosum) and bulbs of snowdrop (Galanthus woronowii Los.), respectively, while huperzine A (HupA) has been isolated from moss (Huperzia serrata (Thunb.Ex Murray) Trev.).HupA is selectively potent and reversible inhibitor with a bet ter therapeutic index than physostigmine and tacrine. 32In fact, HupA is clinically prescribed in China for symptomatic treatment of AD.In spite of their excellent AChEinhibition capacity, the above drugs have serious side effects such as Nausea, vomiting, diarrhea, weight loss, loss of appetite and muscle weakness.As we have found during our analysis, although we use the same data to calculate all these different parameters (%I, IC 50 , V max , K m and K i ), if we do not care about the relationship between parameters the calculation will be wrong and there is a high chance of getting a higher percentage of inhibition.We have searched through literatures but did not find such a study which had examined the plants we studied and given all the kinetic parameters that we calculated.Hence, we could not compare our results with others.
We have compared the IC 50 values for those which have less than 3 µg/µl of IC 50 by Student ttest in Table 3 in order to see how much difference between two IC 50 is considered significant. 27,30For each IC 50 there is a parti cular range to be significant from others, up and down.

Figure 5 :
Figure 5: Lineweaver-Burk plot and Michaelis-Menten plot for Tacrine.Non-Competitive inhibition at two levels.Tacrine was used as a known AChE inhibitor that shows non-competitive inhibition to prove that the assay has been set up correctly.Control (Zero Inhibition), Tcr 01: 16.59 nM and Tcr 02: 33.19 nM.

Table 3 : Student t-test to compare IC 50 values for those which have less than 3 µg/µl
* ) and for α = 0.01: (1 ** ).The table shows the difference not only between two different plants but also between two different solvent for same plant.
and herein V max decreases while K m increases.Two extracts have shown noncompetitive inhibition.Noncompetitive inhibitors bind to enzyme and enzymesubstrate both with equal affinity and K m remains same but V max decreases.Eleven extracts have shown uncompeti tive inhibition in which inhibitor only binds to enzymesubstrate complex.In uncompetitive pattern V max and K m both decrease.And finally, 4 extracts have not given any proper pattern (Table 1/[S] (Lineweaver-Burk plot).The following were the distribution of inhibition patterns: Two extracts had competitive inhibition, 11 extracts had mixed inhibition, 2 extracts had noncompetitive inhibition, Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 substrate both 3,18Many other plant compounds have shown remarkable AChE inhibitory capacity, for example, withanolides from Withania somnifera Dunal.(ashwaganda or Indian ginseng), cur cuminoids from Curcuma longa L., tanshinones from Salvia miltiorrhiza Bunge and quercetin from Quercus sp.(oak) and their full potentials has yet to be investigated.3337Therefore,it seems that plants have a vast kingdom of natural sources of several compounds for combating neurodegenerative diseases.In this study, 30 plant extracts were assayed at two levels of plant extract concentrations, 0.829 µg/µl and 1.037 µg/µl.Two extracts have shown competitive inhibition in which inhibitor only binds to free enzyme and V max remains same but K m increases.Eleven extracts have shown mixed inhibition in which inhibitor can bind to either free enzyme or enzyme their solubility in phosphate buffer.That is why we should use %I only to calculate the IC 50 for each extract independently and then use IC 50 for comparison.
Calculation of the t value and knowing the difference between two extracts can be very useful if we use the extracts for further separation and purification and/ or experiments using cell line or mouse model.It can be helpful when the toxicity and availability of the plant extract is considered.In nutshell, our results demonstrate that selected plants from North East India collected and assayed under the current study contain appreciable amount of AChEinhibitors.It is therefore imperative that more such plants of North East India are screened for potential AChEinhibitors, which could pave the way for the development of new classes of chemical compounds.Currently our work is under way for further separation and assaying of the extract fractions which might provide much better AChEinhibition profile.The comparative study of the isolated fraction against known AChEinhibiting drugs could provide valuable insight for development of newer lead compounds.