Learning, the formation of memory, and the consequent storage and retrieval of individual experiences thus acquired have been the most important aspects of evolution. Learning and memory are considered to be the distinguishing factors that determine the phylogenetic development of the adaptation and have thus continu-ously contributed to the process of the survival of species.
At the individual level, learning and memory are fundamental prerequisites for cognitive functions, emotional reactions, consciousness, speaking, thinking, and the ability to construct internal models of the surrounding milieu within the frame-work of time and space and thus able to take crucial decisions at critical moments. This helps the individuals to shape, broaden, and govern their living space and also to predict the future from the past (Matthies, 1989). Memory is the most important aspect of maintaining the identity and individuality of a personality. Thus, every great civilization had recognized the close correlation between memory and individ-ual personality. In the great book of wisdom Srimad Bha~gwat-Gita~, Lord Krishna has said smriti bhransha~dbudhina~so budhina~satprsyati, meaning memory impair-ment leads to loss of identification and destruction of personality (Goenka, 1967). In Greek mythology, the mother of Muses, the nine Goddesses presiding over arts and science, was Mnemosyne, which literally means remembrance and memory.
Hence, the capacity of the individual organism to learn can be considered as the most important mechanism of adaptation. However, the various theoretical concepts of learning and memory have not been clear on the question of the nature of the performance of memory. This state of affairs seems to stem mainly from the fact that the various definitions of learning have large variation. Let us take the concept of “a relatively permanent change,” which is included in various definitions of learning. Should this requirement imply that all memories are permanent or that some are transient? Various theories of learning are usually silent on this vital ques-tion. There does not seem to be any logic to assume that all consequences of experi-ence are permanent. In fact, there are several overriding reasons to suggest that this cannot be the case. Many of the stimuli surrounding an individual and evoking a specific functional reaction have little or no adaptive significance. It makes very little sense in terms of economy or adaptation that all stimuli should produce enduring consequences in the brain. On the other hand, it is of adaptative value to be able to store long-term representation of experiences that are either particularly meaningful (in terms of consequences) or are frequently repeated.
This plausible theory, supported in the daily experience of life, has been succinctly explained in A Study in Scarlet by Sir Arthur Conan Doyle. Dr. James Watson was astonished that Sherlock Holmes was supremely unaware of the Copernican theory and the composition of the solar system.
“You appear to be astonished” he (Sherlock Holmes) said, smiling at my (Watson’s) expression of surprise. “Now that I do know it I shall do my best to forget it.”
“To forget it!” (Watson)
“You see” he (Holmes) explained, “I consider that a man’s brain originally is like a little empty attic, and you have to stock it with such furniture as you choose. A fool takes in all the lumber of every sort that he comes across, so that the knowledge which might be useful to him gets crowded out, or at best is jumbled up with a lot of other things, so that he has a difficulty in laying his hands upon it. Now the skilful workman is very careful indeed as to what he takes into his brain-attic. He will have nothing but the tools which may help him in doing his work, but of these he has a large assort-ment, and all in the most perfect order. It is a mistake to think that little room has elastic walls and can distend to any extent. Depend upon it there comes a time when for every addition of knowledge you forget something that you knew before. It is of the highest importance, therefore, not to have useless facts elbowing out the useful ones.”
“But the Solar System!” I (Watson) protested.
“What the deuce is to me?” he (Holmes) interrupted impatiently: “you say that we go round the sun. If we went round the moon it would not make a pennyworth of difference to me or to my work.” (emphasis added) (Doyle, 1984)
This prescient description by Doyle bears testimony when we consider the currently accepted concept of executive function. In understanding the executive function, the import of the “working memory” (the ability to hold a goal or relevant information, not allowing any distraction and interference from other sources) has to be considered. The efficiency of executive function is closely related to the converse ability to keep interfering information out of working memory and also out of the set response options. Implicit in this postulation is the ability to withhold or suppress such responses that would become incompatible with the goal when the context changes dynamically. Such ability is also termed as “response suppression” and is emphasized, for instance, in various theories of attention deficit/hyperactivity disor-der (Barkley, 1997; Schachar et al., 1993).
The individual memorizes only those information that are useful in life and that help him to survive. Thus, the ability to suppress unwanted memories and also to avoid distractive memory through executive control, as postulated by several researchers (Anderson and Grew, 2001; Anderson et al., 2004), assumes a very significant importance in efficient functioning of day-to-day life. This is one vital aspect that one has to methodologically evolve to study either the drugs influencing or the various mechanisms underlying the processes of learning and memory in experimental animals or human beings.
Bacopa monniera (Linn) Wettst [Synonyms: Herpestis monniera (Linn) HB&K; Grahola monniera Linn; Monniera cunerfolia Michx (Vernacular: Brahmi)]; family: Scorphulariacae is a perennial herb found throughout India in wet and marshy regions. It is frequently mentioned in the religious, social, and medical trea-tises of India since the time of Vedic civilization. Its antiquity can be traced to the time of the Athar-Ueda (science of well-being) written in 800 BCE. The various treatises of ancient and medieval India have laid emphasis on the ability of bacopa in curing various diseases, particularly its unique ability to improve memory, sharpen intellect, facilitate acquisition of newer information, and promote learning. Because of its unique properties many eminent Indologists (Roy, Chandra, personal commu-nication) and modern researchers (Russo and Borrelli, 2005) believe that the word bra~hmi originates from Lord Brahma~, the creator of the Universe and the originator of Áyurveda in Hindu mythology.
Bacopa is an extremely important plant of the Indian system of medicine, viz., Ayurveda. In the classical Ayurvedic text of Çaraka (Sharma, 2009), bacopa has been classified as medhya-rasa~yan (medhya: memory enhancing and rasa~yan: rejuvenating). Çaraka has described the most unique features of the efficacy of bacopa in alleviating old age and age-related diseases, promoting memory and intellect, enhancing life span, providing nourishment, excellence, clarity of voice, complexion and luster (Sharma, 2009). Çaraka has also described a disease that he has termed as atathvabhe~ne~vesha~m and has also diagnosed it to be the most dreadful mental illness. The symptoms of the disease resemble a combination of hallucination, schizophrenia, obsessive com-pulsive disorder, and severe psychosis (Sharma, 2009) and can be cured only by taking expressed juice of bacopa. The bacopa juice has also been prescribed as cure for epi-lepsy (Sharma, 2009). In another treatise, viz., Sus’ruta-Samhita, it has been mentioned that a 3 week course of bacopa juice will produce photographic memory and a person can retain hundred words uttered only twice daily (Singhal et al., 2009). Bacopa with clarified butter has been prescribed to be highly beneficial for leprosy, intermittent fever, epilepsy, and insanity (Singhal et al., 2009). A bacopa-based drink has been pre-scribed for uroliathiasis (Singhal et al., 2009) and a bacopa-based liquor to cure skin disorder (Singhal et al., 2009).
Because of the traditional importance and unique therapeutic claims of bacopa, it attracted the early attention of chemists in India. Some rudimentary investigations were done earlier by the chemists of Banaras Hindu University (BHU), Varanasi (Bose and Bose, 1931; Sastri et al., 1959). The initial neuropharmacological investi-gations were also done in BHU. Malhotra and Das (1959) reported a sedative effect of glycosides named hersaponins by them. Aithal and Sirsi (1961) found that the alcoholic extract and to a lesser extent the aqueous extract of the whole plant and chlorpromazine improved the performance of rats in motor learning. Sinha (1971) had reported that a single dose of the glycoside hersaponin is better than pentobar-bitone in facilitating acquisition and retention of brightness discrimination reactive.
It is difficult, however, to interpret these results in the context of the known tradi-tional claims of improving learning and memory.
CHEMICAL INVESTIGATIONS
Rastogi and Dhar (1960) at the Central Drug Research Institute (CDRI) were the first to undertake a systematic chemical examination of the plant. The following constitu-ents were isolated (Chatterjee et al., 1963):
Yield (On the Weight of Dry Plant)The activity of the ethanolic extract was traced to a mixture of triterpenoid saponins, designated as bacosides A and B. Both the bacosides showed single spots on TLC on silica gel, while bacoside A was obtained as colorless needles (Chatterjee et al., 1965) and bacoside B was colorless powder (Basu et al., 1967). Bacoside A is levo-rotatory and bacoside B is dextro-rotatory.
Bacoside A was the major component of the plant and comprised two sets of saponins. One set was derived from pseudojujubogenin as the genuine aglycone. The pseudojujubogenin on acid hydrolysis furnished four triterpenoid transforma-tion products, viz., bacogenins A1, A2, A3, and A5 (Kulshreshtha and Rastogi, 1973). The jujubogenin on acid hydrolysis yielded two triterpenoids with triene side chains as transformation products. These triterpenoids were designated as bacogenins A4 (trans) and (cis).
Seasonal Variations
14 months commencing from March. Thus, it covered all the five seasons of India, viz., spring, summer, monsoon, autumn, and winter, taking care of a wide range of temperature and humidity. From this study it was concluded that bacosides A and B are available in May. In the rest of the months, other compounds start appearing and disappearing (Rastogi et al., 1996).
Bacosides-Enriched Standardized Extract of Bacopa
It is essential for plant extracts in order to be therapeutically effective to leave all the constituents intact because the therapeutic effect is generally the result of concerted activity of several active constituents as well as the most of the accompanying substances. Although these inert accompanying substances do not directly affect the therapeutic mechanism, it is reasonable to use the complex mixtures of compo-nents provided by a medicinal plant because these inert components might influence bioavailability and have an optimum effect on the pharmacodynamics and pharma-cokinetics of the active components. Further, inert plant components would augment the stability as well as minimize the possible side effects. If there are different active compounds present in a therapeutically active plant product, they might have addi-tive and potentiating effect (Williamson, 2001). The memory-enhancing effect of bacopa is much more than can be explained by its bacosides contents. Similar obser-vations have been made with several other plants as well (Dhanukar, 1998; Dhawan 1997; Singh et al., 1996). This approach of keeping all the chemical constituents intact has certain inherent advantages, and now, in traditional herbal medicines, such multiple components are purposely combined to provide physiological balance and to harmonize drug effect (Handa, 1998).
Therefore, a standardized extract of bacopa containing a minimum of 55% ± 5% of bacosides with an optimum concentration of bacogenins (especially baco-genin A3) vis-a-vis memory-enhancing effect was developed. This was termed as BESEB CDRI-08.
Further, it becomes imperative to mention the fact that damarene-type- triterpenoid saponins are major constituents of several reputed herbal drugs including ginseng, used for centuries. Jujubogenin glycosides have also been isolated from a number of reputed medicinal plants of rhamnaceae and scrophulariaceae families. However, what is unique about bacopa is that in addition to jujubogenin glycosides, pseudoju-jubogenin glycosides are also present and these have been reported together so far only from this medicinal plant (Garai et al., 1996; Russo and Borrelli, 2005).
If Rastogi and coworkers at CDRI were the first to undertake a systematic chemical examination of bacopa, Singh and Shankar (1996) also of the CDRI, were the first to elucidate a targeted memory-enhancing effect of the bacosides-enriched standard-ized extract of B. monniera (BESEB CDRI-08).
Certain Methodological Considerations
The preparation, discovery, research, and development of drugs with psychological effects, in general, and memory effects, in particular, have occupied the interest and energy of humans since the inception of civilization. The experiments of Lashley (1917) demonstrating the facilitation of maze learning by low dose of strychnine sulfate is perhaps the first recorded scientific investigation on a drug affecting learn-ing and memory. Ever since then, there has been a tremendous upsurge in the study of drugs influencing learning and memory. With a greater widening of horizon, tech-nological advancement of laboratory research equipments, easy availability of target pharmacological tools, the refinement of conceptual framework has also signifi-cantly improved. All these have resulted in the study of drugs facilitating, impairing, and neuromodulating learning and memory in greater depth and detail.
However, it will not be wise to overlook the fact that the evaluation of a new drug affecting learning and memory is still beset with several methodological problems. Even though these have been dealt in detail elsewhere (Singh and Dhawan, 1992), it would still be pertinent to summarize some of the overriding issues.
At the outset, one important issue is to distinguish the drug effect on learning vis-a-vis its effect on performance. A performance in the absence of a reward or motivation would only reflect latent learning. A true measure of the actual learning can only be witnessed when the experimental animals are motivated or when the reward is introduced (Blodget, 1929).
It is also important to dissociate the effect of a drug under the conditions when the learning by the experimental animals takes place in the drugged state. Such learning performance is not reflected during the normal state when the influence of the drug wears off. Generally, any drug having a depressant effect, for example, chlordiaz-epoxide, chlorpromazine, and pentobarbital, will produce such dissociation effects.
Similarly, it is of great theoretical significance to consider that the effect of a given drug on learning and memory will also depend upon the fact whether the primary action of the drugs is on the receptor or effector systems. Any drug having only peripheral effects will still indirectly alter the CNS function by modifying the inputs to the CNS. Conversely, any centrally acting drug will also alter the functions of peripheral nervous system.
The type of apparatus chosen to study the effect of a drug on learning and memory is also a serious consideration. Small differences in the design of a maze can produce discrepant results in the studies of the effects of drugs on learning. For example, the addition of retracing doors has been shown to increase the reliability of mazes (Silverman, 1978).
Certain Conceptual Considerations
Apart from these methodological considerations, our endeavor to formulate a strategy to elucidate the memory-enhancing effect of BESEB was also substantially influenced by different conceptual and experimental postulates available in the 1970s. If the classical idea of Ivan Petrovich Pavlov about the formation of temporary connection provided an excellent starting point, the hypothesis of Hebb (1949) of convergence of pathways and the coactivity of neurons resulting in modifiable synapses provided the additional fillip. An extremely important consideration was the result obtained by McGaugh (1966), who taking a cue from the consolidation hypothesis evolved from the studies of electroconvulsive shock (ECS) induced amnesia (Dunccan, 1949; Glickman, 1961), suggested that the memory formation is a time-dependent process.
The experimental demonstration that ECS intervention was effective only when applied immediately after the completion of a learned task and was found to be inef-fective if the time lapse between completion of the learned task and application of ECS exceeded even 15 min was considered to be of great significance in determining the time of drug application to influence consolidation. Generally, a drug should be applied immediately after the training, but the fact that pretraining applications are also known to selectively influence consolidation cannot be overlooked. Pretraining application will thus have a biphasic effect both on the acquisition and the consolida-tion processes. However, there have been certain notable exceptions. For instance, it has been reported that naloxone enhanced the consolidation of Morris Water Maze learning with pretraining but not with post-training application (Decker and McGaugh, 1989). The nootropic CGS5649B when administered immediately after training failed to improve the task performance in maze learning, but it significantly enhanced task performance when administered 8 or 24 h after training (Mondadori, 1990).
Another important consideration is that short term memory (STM) does not con-solidate into long term memory (LTM) in a linear curve manner. This has been sug-gested by the classical studies of the German Scientist Herman Ebbinghaus at the end of the eighteenth century and has been supported by the investigations of Müller and Pilzecker (1900).
These researches have demonstrated that during the process of consolidation when information was stored in STM, the consolidation of long-term trace entailed certain time-dependent processes. As stated earlier, they also found that during STM and consolidation of LTM, the memory trace was very sensitive to interventions, but when the memory was consolidated, it became stable and hence could not be influ-enced by any intervention. Later, the time course of both STM and the consolidation of LTM were more precisely elucidated by several investigations (Agranoff et al., 1967; Baronodes and Cohen, 1967; Bennett et al., 1977; Duncan, 1949; Flexner et al., 1963; Flood et al., 1986; McGaugh, 1966). However, the investigations of Kamin (1957, 1963) have indicated that there are peak and trough in the retention curve during the course of LTM. These observations have led to the hypothesis that mem-ory formation takes place in three or four stages, which has been substantiated by corresponding experimental data (Gibbs and Ng, 1976; Frieder and Allweis, 1982; Matthies, 1974; Ott and Matthies, 1978).
Learning Models
However, the differences in the results obtained in the time course of consolidation, on different memory stages as well as the duration of amnesic effects, can only be accounted for by considering the following two factors: first, the stimuli applied in different experimental tasks were of different intensities; second, the signals thus converge at different levels of functional hierarchy.
This is of great methodological significance and has influenced our choice of a suit-able learning model to study the effect of BESEB or learning and memory. We also, in the first instance, rejected learning at a relatively low level of complexity and neu-ronal organization, for example, an one trial passive avoidance task, even though this offers the advantage of rapid acquisition. In order to obtain a confirmatory memory-enhancing effect, our choice was a model that should induce well-distributed and extensive cellular changes during the learning discrimination task and that would also involve a reversal of innate behavior. Our first choice was a foot-shock-motivated brightness discrimination task on rats in a semiautomatic Y-maze as the standard procedure in most of the studies done on the elucidation of the memory-enhancing effect of BESEB CDRI-08.
Punished by foot shock in the dark alley
This method has been described in detail by Ott et al. (1972), Singh et al. (1975), and Ott (1977). Briefly, rats were trained to escape a foot shock by running into one of the other two arms. The entry into the dark alley was punished by a foot shock (1 mA). For the next trial, the former goal box was used as a start box, which also helped in avoiding handling during the training session. The intertrial inter-val was approximately 60 s. The direction of alley illumination was changed after every three trials to avoid position discriminative. The training consisted of 40 trials (45 min). The retention was tested 24 h after training using the same paradigm as in the learning session. The number of negative trials, i.e., the number of foot shocks received during training (Ts) and relearning (Rs), were used to calculate percent sav-ing, which was the difference between the two, expressed as percent of the former:
100
% savings : Ts - Rs × TSThe results obtained from the relearning test was also analyzed on the basis of increase in positive response during relearning (SR), i.e., the difference between RR (positive response during relearning) and TR (positive response during training). A response was considered to be positive when the rat ran immediately into the lighted alley in the last run prior to and the first run after the change in the direction of alley illumination.
In this training procedure, both the percent saving and SR appear to be less depen-dent on influences of performance than is true of latency measures. However, it is also important to use a battery of diversified tests whenever attempting to measure the effect of any drug on level of motivation or emotionality (Miller and Teuber, 1968). Therefore, we decided to use different test models for the evaluation of the memory-enhancing effects of BESEB CDRI-08. The battery of tests used consisted of positive (reward) as well negative reinforcement (punishment), labile phase of memory (when the memory is in formative stage), and stable phase of memory (when the memory formation has taken place). Moreover, the training should be such that it allows a demarcation in three phases of memory, i.e., acquisition, consolidation, and retention. The model should also clearly distinguish between the successive stages of short-term, intermediate, and long-term memories (T. Ott and H. Matthies, personal discussion, 1975). The foot-shock-motivated brightness discrimination reaction fulfilled most of these criteria. However, it was a training with negative reinforcement (foot shock), the training was completed in one session, and the memory formation was labile. The active avoidance, Sidman’s conditioned avoidance, and conditioned taste aversion responses were initially used to confirm the memory enhancing effect of CDRI-08. Later, one-trial passive avoidance response in few experiments were used.
Other Learning Models
In the active conditioned avoidance response, which was a modified method of Cook and Weidley (1957), the rats had to jump on a wooden pole to avoid a foot shock. The method was based upon the hypothesis expounded by many neurobehavioral scientists that learning is essentially of two types, viz., procedural learning and declarative learning.
In the procedural learning, the animals have to learn that presentation of an unconditioned stimulus (US) is preceded by conditioned stimulus (CS). In other words, they learn that a particular response or an event (presentation of US in the form of a foot shock of 1 mA intensified for a duration of 15 s) is reinforced when a discriminative signal or CS (a buzzer presented for 15 s) is given. Thus, the active conditioned avoidance response essentially consists of a series of actions conducted in a certain order and manner that ultimately computes into a subroutine.
A well-trained animal not only shows procedural learning but also exhibits declar-ative learning by responding to CS. In few cases, animals attain a very high level of training by exhibiting spontaneous learning and respond to the milieu or environ-ment (secondary conditioned stimulus [SCS]) without waiting for the CS or the US.
The third learning schedule was the continuous avoidance response as evolved by Sidman (1953, 1956). It is a relatively complicated task for rats, as no contingencies between avoidance behavior and exteroceptive stimulus (e.g., a tone as conditioned stimulus) are involved. The training comprised a daily session of 1 h with lever pressing selected as the avoidance response. The programming is done in such a manner that each lever press delayed the shock for 25 s. A minimum interval of 15 s was assured between two successive shocks, i.e., when no avoidance behavior occurred. However, when the avoidance behavior occurred, i.e., when the lever was pressed, the shock was further delayed by 10 s. In other words the next shock was delivered after 25 s. Thus, the shock–shock interval was 15 s and shock–response interval was 25 s (SS15RS25). A trained animal responded repeatedly by pressing the lever at a stationary rate to avoid shocks (Kuribara et al., 1975, 1976).
The three learning schedules employed in these experiments consisted of a nega-tive reinforcement, i.e., a foot shock to motivate the animals. Hence, a fourth confir-matory test involving positive reinforcement, i.e., reward in the form of conditioned taste aversion (CTA) response was performed.
One of the most important tasks of neuroethology is to develop laboratory versions of ethological experiments that would make it possible to investigate the underlying brain mechanisms. Particularly important in this respect are the neural processes controlling food intake and licking or aversion to some items of food. In the CTA response, single male rats were kept in individual cages containing openings for two 30 mL Richter tubes with 0.5 mL gradations. The rats had access to standard food pellets but were deprived of water overnight. Each rat was allowed to drink tap water from the Richter tube for 1 h only every morning. After a few exposures, all rats started drinking after presentation.
These rats were then tested in gustatory discrimination apparatus by exposing them simultaneously to two drink spouts: one delivering sucrose, the other contain-ing an aversive fluid Lithium Chloride (LiCl). The CTA test is based on the capabil-ity of rats to avoid a novel food or liquid, the ingestion of which leads to undesirable side effects (moderate gastrointestinal disorder) (Barker et al., 1977; Garcia et al., 1955; Milfram et al., 1977; Rozin and Kalat 1971). A trained rat rapidly finds the sucrose-containing spout and starts drinking there from. After the stabilization of licking sucrose is suddenly replaced by the aversive LiCl fluid. The position of the spouts is changed through motarized rotation by 180°. An important prerequisite of stationary retrieval condition is that prolonged testing does not cause CTA extinc-tion. High motivation of the animals to avoid the aversive concentrations eliciting moderate gastrointestinal disorder after the ingestion of a few milliliters of isotonic solution of LiCl (0.15 M) can serve as a combined CS (salty taste) and US (symptom of gastrointestinal disorders).
Animals
The choice of experimental animals were either inbred albino Charles Foster or Sprague Dawley rats, which because of their similarity with human neuromorpho-logical, neurophysiological, and neurochemical parameters are considered to be excellent infrahumans.
Dose Schedule
The dose schedules of BESEB CDRI-08 were chosen after careful considerations (Pandey, personal discussion, 1976). The best course to determine the dosage sched-ule was to perform a series of preliminary experiments with daily oral administration of different doses for varying number of days. It was established that the administration of 40 mg/kg p.o. for 3 days was optimal in producing a significant effect.
However, the doses, frequency of oral administration, and training–relearning intervals varied according to the experimental design, schedule, and level of motivation.
The dose regimen for CTA is summarized in Table 1
Summary of Results
The results of these investigations confirm that BESEB CDRI-08 has facilitatory effect on memory and learning in a wide variety of responses. The effect of BESEB CDRI-08 is manifest both in negative reinforcement (shock-motivated brightness discrimination reaction, conditioned and continuous avoidance responses) as well as positive reinforcement (conditioned taste aversion). This is significant as BESEB CDRI-08 facilitated the responses that are susceptible to the effects of punishment as well as reward. BESEB CDRI-08 is thus markedly effective in increasing the memory in a wide variety of responses. (Singh and Dhawan, 1978, 1982; 1974a,b; Singh et al., 1990a,b, 1996a,b, 1988).
TABLE 1
Summary of Dosage Regimen for CTA
Group Treatment Dose (mg/kg po)
I Saline Saline 0.5 mL
II BESEB-CDRI-08 2.5
III BESEB-CDRI-08 5.0
IV BESEB-CDRI-08 7.5
V BESEB-CDRI-08 5.0a
a This group was not intubated with Li CI on day 1.
A feature of memory formation across the animal kingdom is its progression from short-lived labile form to a long-lasting stable form. But as already described earlier, memory formation is not a direct flow of neuronal activity from short-term to long-term storage. Congruent lines of evidences have pointed instead to an intricate, multiphasic pathway of consolidation. Based on their experimental investigations, Ott and Matthies (1978) have postulated that memory essentially exists in two forms, viz., short term and long term. Both these forms start simultaneously and a deficit in retention curve is obtained at a point where these two forms overlap, i.e., it is V-shaped.
The hypothesis was tested in the foot-shock-motivated brightness discrimination reaction in the Y-maze and it was found that the retention curve instead of being V-shaped was W-shaped, i.e., there were deficits occurring at two points, viz., 1.5 h and 4.0 training–relearning intervals. Therefore, it was presumed that, at least in this experimental model, there are perhaps three forms of memory, i.e., short term (few seconds to minutes) and long term (few hours to days) and in between there exists an intermediate form of memory (few minutes to hours). The two deficits apparently occur at points where one memory overlaps with the other memory.
The BESEB CDRI-08 in a dose of 20 mg/kg p.o. when given 30 min prior to training in a shock-motivated brightness discrimination reaction abolished these deficits when relearning was done at various time intervals after training. A support dose of 10 mg/kg p.o. given 30 min prior to the 24 h relearning test also produced a significant enhancement in the relearning index. These results suggest that the facilitatory effect of BESEB CDRI-08 is apparently due to their ability to consolidate the reten-tion at the earliest form, i.e., short-term memory.
During the period of consolidation, memory can be disrupted through administra-tion of a wide variety of amnesic agents. ECS, hypothermia, and hypoxia are some of the noninvasive procedures that induce retrograde amnesia. While most of the amnesic agents used induce retrograde amnesia, there are others that cause temporary amnesia. For instance, diethylthiocarbamate, an inhibitor of synthesis of noradrenaline, disrupts intermediate memory when administered prior to training. Similarly, hypoxia disrupts intermediate memory specifically when administered immediately after training. Therefore, it appears that memory consolidation involves both serial and parallel processing of information.
In order to have comparable information, the experimental protocols were kept uniform, and retrograde amnesia was produced in rats by three interventions, viz., immobilization stress administered for 17 h, ECS (0.5 mA, 50 Hz, 0.5 s) administration and scopolamine. All these interventions were administered immediately after the completion of training. The training schedule used was a brightness discrimination reaction. BESEB CDRI-08 used was in a dose of 20 mg/kg p.o. × 3 day. The amnesic treatments led to a significant disruption of consolidation in control groups, as measured by percent savings in the relearning test performed 24 h after the completion of training. The BESEB CDRI-08 pretreatment significantly attenuated the amnesic effects in all these treatments (Singh and Dhawan 1997, 1998).
A qualitative similar effect was reported by Sethy et al. from Upjohn Company, Kalamazoo, MI. BESEB CDRI-08 attenuated the scopolamine-induced amnesia (Sethi et al., 1994).
Another investigation was done by Manjrekar (1996) on the effect of CDRI-08 on scopolamine-induced amnesia in rats and mice employing elevated plus maze test, step-down passive avoidance test, and Cook and Weidley pole-jumping avoid-ance test. It was found that a single dose of 10 mg/kg BESEB CDRI-08 to be either equipotent to or more effective than similarly administered piracetam (150 mg/kg), cyclandelate (200 mg/kg) or Gingkgo biloba and Withania somnifera extracts (100 mg/kg p.o. for 4 days) in attenuating the amnesic effect of scopolamine. This finding was consistent for all the three test models used.
Das et al., (2002) have also found BESEB CDRI-08 to be equipotent to Ginkgo biloba extract in counteracting the scopolamine-induced transient amnesia on trans-fer latency time.
As can be evidenced, Ginkgo biloba and BESEB CDRI-08 have qualitatively comparative effect on memory. The insignificance difference can be attributed to suppression of fear induced by BESEB CDRI-08 because of its anxiolytic effect (Shanker and Singh 2000). The Ginkgo biloba extract has not been reported to possess any anxiolytic property.
Prabhakar et al. (2007) and Saraf (2009) also evaluated the effect of BESEB CDRI-08 on diazepam-induced amnesia in mice in Morns Water Maze. Benzodiazepines are known to produce amnesia through the involvement of GABA ergic system and hence the reversal of memory deficit induced by diazepam was investigated.
The results revealed antiamnesic effect of CDRI-08 (120 mg/kg p.o. administered-istered 60 min prior to training) on diazepam (1.75 mg/kg p.o. administered 30 min prior to training)-induced amnesia. The degree of reversal by BESEB CDRI-08 was significant as it progressively reduced escape latency time when mice treated with diazepam were subjected to acquisition trials 7 days after train-ing. The results suggest that the antiamnesic effects of BESEB CDRI-08 follow a gamma-aminobulyric acid—benzodiazepine pathway, possibly affecting long-term potentiation.
Singh et al. (1979a,b) and Singh and Singh (1980) were the first to suggest an anti-anxiety effect of a bacopa extract based on its effect on gross behavior and prolon-gation of barbiturate hypnosis in rats. Shanker and Singh (2000) have also reported anxiolytic activity of BESEB CDRI-08 by observing the effect on prolongation of barbiturate-induced sleeping time, aggregation of amphetamine hyperactivity, inhibition of clonidine-induced fighting and biting behavior, and increased sur-vival under hypoxia. The BESEB CDRI-08 also has a mild antidepressant activity as evidenced by the reversal of reserpine-induced syndromes and the enhancement of immobility time in swim despair test in mice (Singh et al., 1996b). It has also been found that BESEB CDRI-08 protects metrazol-induced convulsions both in multiple (ED50: 216 mg/kg p.o. × 5 day) and single (ED50: 647 mg/kg p.o.) doses in mice (Singh et al., 1996a,b). Although there is no clinical evidence available, on the strength of other supportive data it can be postulated that BESEB CDRI-08 may alleviate epilepsy by its ability to stabilize seizures (electrical activities) in the (cell membrane of) brain.
In recent times, adaptation has emerged as a unique concept as it helps living beings to combat day-to-day stress and is therefore considered to be essential for survival.
Experimental animals were exposed to two types of stress, i.e., acute and etronic. In acute stress, rats were confined for 150 min inside an acrylic hemicylindrical plastics tube for a day, whereas in chronic stress the confinement was for the same duration but for 7 consecutive days. The BESEB CDRI-08 (80 mg/kg p.o. × 3 day) exhibited pronounced adaptogenic effect by normalizing the changes induced by stress, e.g., prevented the formation of ulcer, reversed adrenal and thymus hyper-trophy, spleen hypotrophy, normalized blood sugar, and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) activities. A com-parative study was made with Panax quinquefolium (PQ), one of the recognized varieties of ginseng (Rey, 2009). PQ produced similar effect except that unlike BESEB CDRI-08, PQ failed to reverse the formation of ulcer, adrenal hypertrophy, and hyperglycemia even at a higher dose of 100 mg/kg p.o. × 3 day (Rai et al., 2003).
These findings were further confirmed by Sheikh et al. (2007) by investigating the effect of CDRI-08 on stress-induced changes in brain corticosterone and brain monoamines in rats. The acute stress was the same as in the study of Rai et al. (2003), but instead of chronic stress, a chronic unpredictable stress (CUS) was administered. The CUS regimen involved exposing two different stressors of variable intensity every day in an unpredictable manner for seven consecutive days. These stresses include immobilization (150 min), forced swimming (20 min), overnight soiled cage bedding, foot shock (2 mA for 20 min), day–night reversal, and fasting (12 h). The salient findings of this study are that BESEB CDRI-08 normalized the stress-induced alterations in plasma corticosterones and levels of monamines noradrenaline, sero-tonin, and dopamine both in the cortex and hippocampus regions of the brain, both of which are more vulnerable to stressful conditions.
The importance of this finding lies in the fact that changes in monaminergic activity result in behavioral changes as well as a cascade of hormonal release from hypothalamus–pituitary–adrenal (HPA) axis. These abnormal alterations in mono-amines under prolonged stress has been associated with a wide range of central and peripheral disorders like depression, anxiety, drug abuse, obsessive compul-sive disorder, eating and sleeping disorders, hyperglycemia, and decreased immune response. The present-day lifestyle accompanied by the advent of various stress-related disorders entails unduly heavy physiological and psychological demands. It is widely felt by health planners that there is an urgent need to develop agents to overcome these abnormalities. The BESEB CDRI-08 has thus the required potential to fulfill this demand.
The antistress effect of CDRI-08 was studied at doses of 20 and 40 mg/kg p.o. administered for seven consecutive days in rats (Kar Chowdhuri et al., 2002). For administering the stress, the modified method of Ramchandran et al. (1990) was used. Briefly, rats were fasted overnight and subjected to a multiple stress of restraint, cold (5°C), and low-oxygen tension (428 mm Hg pressure, equivalent to an altitude of 15,000 ft) in a vertical decompression chamber. Under such stressful conditions, the core temperature of the experimental animals starts falling. The treatment is termi-nated when a core temperature of 23° ± 1°C is attained. The modulatory effects of BESEB CDRI-08 on stress-induced changes in expression of HSP70 and activities of superoxide dismutase (SOD) and cytochrome P450-dependent 7-pentoxyresorufin-D-alkylase (PROD) and 7-ethoxy-resorufin-0-deethylase (EROD) were estimated. The data clearly demonstrated the potential of BESEB CDRI-08 in reducing stress by modulating the expression of Hsp70 and the activities of P450s and SOD, the enzymes known to be involved in the production and scavenging of reactive oxygen species in different brain regions of the brain.
Amar Jyoti et al. (2006, 2007) investigated the neuroprotective role of the BESEB CDRI-08 against aluminium-induced oxidative stress in the hippocampus and cere-bral cortex of rat brain and compared with L-deprenyl (seleginine). L-Deprenyl is an MAO-B inhibitor and neuroprotectant offering protection against the effects of neu-rotoxins and excitatory amino acids (Ebadi et al., 2002). It is also used as a therapeu-tic agent for the neurodegenerative disorder Parkinson’s disease involving oxidative stress. L-Deprenyl prevents the apoptosis of dopaminergic neurons associated with Parkinson’s disease by altering the expression of a number of genes such as SOD, Bcl-2, Bcl-XL, NOS, CJUN (Ebadi and Sharma, 2003). L-Deprenyl is also a very well-known antioxidant (Xu et al., 1993) and is postulated to have antiaging effects as it has shown to properly extend life expectancy (Bickford et al., 1997).
The data generated clearly indicated that coadministration of BESEB CDRI-08 together with aluminium treatment prevented the latter’s oxidative stress effects. Aluminium reduced superoxide dismutase (SOD) activity significantly. CDRI-08 attenuated this reduction and restored the SOD activity to near normal. Similarly, CDRI-08 prevented the aluminium-induced increase in thio-barbituric acid-reactive substance (TBA-RS) and carbonyls. This protective role is further supported by the microscopic observations, which showed that BESEB CDRI-08 prevented the aluminium-induced lipofuscin accumulation and ultrastructural changes in the hip-pocampal CA1 field. The hippocampal CA1 field was selected for microscopic stud-ies as its pyramidal neurons are potentially more vulnerable to aluminium-induced neurotoxicity (Sreekumaran et al., 2003) and hypoxia (Kawasaki et al., 1990) than CA2 and CA3 hippocampal fields.
The antioxidative effect of the BESEB CDRI-08 was found to be similar to L-deprenyl. This is an important finding as oxidative stress (lipid peroxidation, lipo-fuscin accumulation, etc.) is postulated to be an extremely important contributory component of the ageing process. Because of its antilipidperoxidative and antili-pofuscinogenitric effects, the BESEB CDRI-08 can thus be considered as a poten-tial antiaging substance. This postulation is further strengthened by the similarity between the effects of the BESEB CDRI-08 with those of L-deprenyl, which itself is considered to be a candidate antiaging drug. This is a further indication of the antiaging potential of the BESEB CDRI-08. The importance of this finding can be further gauged by the fact that neuroprotective effects are observed both in hippocampus and cortex. The severity of aluminium induced oxidative stress is more pronounced in the hippocampus and neocrotex regions than any other area of the central nervous system. Such oxidative damage during aging and oxidative stress is postulated to significantly contribute to the impairment of cognitive functions like learning and memory (Fukui et al., 2000). On the strength of these findings the authors have concluded that BESEB CDRI-08, because of its antilipidperoxidative and antilipofuscinogenistic effects, could be considered as a potential antiaging sub-stance (Kaur et al., 2003).
In order to study the mechanism of action, investigations were designed to (1) estab-lish the biochemical correlates of the memory-enhancing effects of BESEB CDRI-08 and (2) to elucidate its cellular mechanism of action.
Biochemical investigations showed that BESEB CDRI-08 enhanced the protein kinase activity in hippocampus. It also induced an increase in protein and sero-tonin and lowered the epinephrine levels in hippocampus. Similar changes were also observed in hypothalamus and cerebral cortex (Singh and Dhawan, 1997). The enhancements of protein and serotonin, and the depletion of norepinephrine levels are indicative of the facilitatory effect of BESEB CDRI-08 on long-term and inter-mediate forms of memory (Angers et al., 1998; Byrne and Kandel, 1996; Cohen et al., 2003; Crow et al., 2001; Kandel, 1987; Kandel et al., 1986; Menaces, 1995, 2003).
In brain tissue, nitric oxide causes activation of the soluble guanyl cyclase enzyme, resulting in an increase in the cellular levels of cyclic GMP (cGMP). A common stimulus for nitric oxide is the activation of a particular class of receptors for the excitatory neuro transmitter glutamate, i.e., the NMDA receptors.
Investigations were done at the Wolfson Institute of Biomedical Research University College, London, to study (1) an effect of CDRI-08 itself to indicate whether it has the ability per se to release nitric oxide and (2) an effect of CDRI-08 in the presence of a submaximally effective concentration of NMDA to elucidate a potentiating or inhibiting effect or whether it simulates nitric oxide formation. The results obtained showed that although CDRI-08 did not possess an intrinsic effect on nitric oxide release, it potentiated NMDA-mediated nitric oxide and cGMP produc-tion in the cerebellum at a concentration of 100 µg/mL.
The hippocampus is a brain area associated with memory formation. At the cellu-lar level, memory is considered to be stored in the form of long-lasting changes in the strength of the synaptic connections between neurons. Such changes in hippocampus are induced through tetanization, i.e., brief stimulation of synaptic connectivity at high frequency.
Using electrophysiological recording techniques, the effect of BESEB CDRI-08 on synaptic transmission in rat hippocampal slices were also studied at the Wolfson Institute of Biomedical Research, University College, London (1997).
The investigations showed that BESEB CDRI-08 possessed clear intrinsic bio-logical effects on synaptic transmission in the hippocampus by producing a synaptic depression during application. This depression reverted to an enduring potentiation when the BESEB CDRI-08 was washout. The changes thus observed are similar to those produced by nitric oxide under identical conditions. Such a change is identified as a natural neuroprotective device.
The result obtained clearly indicated that BESEB CDRI-08 in a concentration of 10 µg/mL was able to change the properties of the hippocampal synapses in such a way that a weak stimulus (which normally has little or no lasting effect) was able to elicit an enduring potentiation. This result has a direct relevance to the memory-enhancing effect of BESEB CDRI-08 (Garthwaite, 1997).
The LD50 of the BESEB CDRI-08 was determined in rats and mice by both oral and i.p. routes. It was >3 g/kg body weight by oral route in both the species, but by i.p. route it was much lower and was found to be 205 mg/kg [with the confidence limit (CL) ranging from 230 to 182] in rats and 224 mg/kg (CL 260–193) in mice.
In the gross observations, the BESEB CDRI-08 (500 mg/kg p.o.) did not produce any significant alterations in the total, ambulatory, and stereotype behaviors in India. The product produced no effect on respiration and undesirable effects like writh-ing, ataxia, tremor, convulsions, etc. were absent. It did not produce any blockade in posture and tones and the eye movements were normal. The pineal, corneal, and righting reflexes were normal, and there was no effect on other reflexes. BESEB CDRI-08 did not produce any neurological deficit and all the innate and motivated behavioral responses were intact. The BESEB CDRI-08 at a dose of 50 mg/kg p.o. did not show anti-inflammatory activity nor produced any change in blood pres-sure, heart rate, respiration, or nictitating membrane contraction in rats. Standard vasopressor response of adrenaline (2–4 µg) and vasopressor responses of acetyl-choline (1–2 µg) were unaltered. The BESEB CDRI-08 at a dose of 100 mg/kg p.o. did not produce any significant diuretic or antidiuretic effect and no fall from a rota rod in a forced locomotor activity test. Graded concentrations of BESEB CDRI-08 (10 and 50 µg/mL) produced an inhibition of serotonin-induced contraction in iso-lated guinea pig ileum preparation (Singh and Shanker, 1996).
These investigations were done in the Division of Toxicology, CDRI.
BESEB CDRI-08 was given once daily orally for 90 days at the doses of 50, 100, and 200 times the effective dose of 40 mg/kg/p.o./day to rats of Charles Foster strain divided into three groups, each consisting of 15 male and 15 female animals. A fourth group of 15 male and 15 female animals were fed with corresponding volumes of vehicle and served as controls. Weekly monitoring of body weights of all the animals was done. Hemograms and urine analysis parameters of the animals were recorded initially and then at monthly intervals of the study. All the animals were sacrificed at the end of 90 days of the study, and terminal blood biochemistry and histopathology of all the important organs and tissues were studied.
Animals continued to remain active and healthy throughout the period of experi-mentation. The treated and control animals gained body weight well in comparison to one another. The laboratory investigations showed no indication of treatment-induced damage in the various hematological and biochemical, parameters, organ weights (both absolute and relative of important organs), and histopathological examinations.
BESEB CDRI-08 was thus found to be safe in rats in the 90-day subacute toxicity by oral route at the aforementioned dose levels.
BESEB CDRI-08 suspended in 1% aqueous gum acacia was given daily by oral route at the doses of 25, 50, and 100 times the effective dose of 10 mg/kg/p.o./day body weight in a single bolus to rhesus monkeys divided into three groups, each consisting of three male and three female animals. Another group of three male and three female animals were given corresponding volumes of 1% aqueous gum acacia alone in a similar manner and served as control. The treatment continued for 3 months. Average 24 h food consumption of the animals was recorded ini-tially and then at weekly intervals throughout the period of the study. Body weights of the animals were recorded initially and then at monthly intervals. Hemograms, urinalysis, and serum biochemistry were done on weeks 0, 5, 9, and 13 of the study. All the animals were sacrificed at the end of the study and bone marrow was examined. The histopathology of all the important organs and tis-sues was studied.
The animals continued to remain active and healthy throughout the duration of treatment. Animals of both drug-treated and control groups showed irregular trends of gain in body weight. The laboratory investigations showed no indication of drug-induced damage in urinalysis and hematological and serum biochemical parameters. Elevations in SGPT (ALT) levels of few animals of the treated group were noted. But there was no functional abnormality in the liver as evidenced by constantly normal levels (treated vs. controls) of serum bilirubin, serum albumin, total serum protein, and prothrombin time estimations. Moreover, there was no evidence of intrahepatic biliary stasis as the levels of serum alkaline phosphatase remained within the ranges of normalcy in the treated groups of animals throughout the study. The autopsy studies (including absolute and relative weights of important organs) and gross and histopathological examinations did not reveal any sign of target organ toxicity.
BESEB CDRI-08 was thus found to be safe in rhesus monkeys in a 3 month sub-acute toxicity study by oral route at the aforementioned dose levels (Srivastava and Sudhir, 1996).
The purpose of the study was to delineate the harmful effects of BESEB CDRI-08 on pregnancy and unborn offsprings with the intention to provide information on the potential hazards to the developing fetus that may arise due to administration of the compound to the pregnant rats (Charles Foster) and rabbits (New Zealand white) by oral route during their major period of organogenesis, i.e., from day 6 to 15 and day 6 to 18 of the pregnancy period, respectively.
Pathogen-free male rats (175–250 g) and rabbits (1.75–2.25 kg) of proven fertil-ity and sexually mature, randomly chosen nulliparous female rats (150–225 g) and rabbits (1.75–2.00 kg) were obtained from the National Laboratory Animal Center (NLAC) of the CDRI. A total of 10 female and 5 male rats and 2 male and 2 female rabbits were used for the study.
Each rat received a daily oral low dose of 50 mg/kg and high oral dose of 100 mg/kg and each rabbit received a daily oral low dose of 26.66 mg/kg and high oral dose of 53.32 mg/kg
Female test animals of timed pregnancy were treated with the test substance daily throughout the appropriate treatment period, i.e., from day 6 to 15 of gestation in rats and day 6 to 18 of gestation in rabbits. Signs of toxicity were recorded as and when they were observed, indicating the time of onset, the degree, and the duration. Females showing signs of abortion or premature delivery were sacrificed and sub-jected to thorough macroscopic examination. The posttreatment observation period continued till 1 day prior to term. During the treatment and observation period cage side observations included changes in skin and eye and mucus membrane, as well as condition of orifices, behavior pattern, etc. Food and water consumption were also observed every week if they were satisfactory or not (in the case of rabbits, only food consumption was measured). Animals were also weighed.
Standard parameters like general signs, mortalities/morbidity, food and water con-sumption, and body weight were observed for parent animal and gross, visceral, and skeletal anomalies (minor differences from normal that were detected either in gross and visceral or in skeletal examinations were observed. The percent post-implantation loss was calculated as the percentage of difference between total num-ber of implantation and total number of fetuses.
The results of parent animals revealed that all animals remained healthy during the period of study and there was no obvious change or any sign of reaction to drug in any of the treated rats or rabbits. No mortality was observed and food and water consumption were normal. A steady gain in weight was recorded among all the rats and rabbits of all the groups. The number of Corpora lutea and implantations were comparable and no intrauterine death (still birth) occurred. The litter sizes of all the groups were comparable and well within the range of normal limit.
The fetal abnormalities observed in treated and control groups were all incidental and have been commonly reported upto 10% even among normal rats and rabbits under normal conditions. The incidences observed were not dose dependent, thus ruling out any correlation with BESEB CDRI-08.
In the gross fetal abnormalities, only 2 out of 128 fetuses (1.56%) of control group, 2 out of 130 fetuses (1.53%), and 3 out of 141 fetuses (2.12%) revealed exter-nal anomalies. In rabbits, the total incidences among the examined fetuses were 1/56 (1.53%) in control group, 0/29 (-) and 0/25 (-) of low and high doses of BESEB CDRI-08, respectively.
The visceral and skeletal examinations of the fetuses showed no significant anom-alies in any of the groups in all the animals.
Therefore, it is concluded that oral administration of BESEB CDRI-08 at the dose levels of 50 and 100 mg/kg in rats and 26.66 and 53.22 mg/kg in rabbits, during their major organogenesis, has not revealed any teratogenic effects (Srivastava and Sudhir, 1996).
Sister-chromated exchange (SCE) and chromosome aberrations (CAs) are the important cytogenetic endpoints used extensively to study the genotoxic effects of drugs. In the pres-ent study, SCE and CA were carried out after in vivo exposure of BESEB CDRI-08 in bone marrow cells of Swiss albino mice of both sexes 10–12 weeks old and weighing 30 g.
In the study, 5-bromo deoxyuridine (BrdU) tablets (50 mg), Boehrringer Mannheim Biochemicals, Colchicines and Cyclophosphami de, Sigma Chemical Company, St. Louis, Mitomycin C, Andrew, Milwaukee, besides BESEB CDRI-08 were used.
For CA analysis, three doses (20, 40, and 80 mg/kg) of BESEB
CDRI-08 were suspended in distilled water and injected i.p. in a volume of 100 µL per mouse. Five animals (three males and two females) were used for each group and also for the control. Negative control mice were injected with an equal volume of distilled water (100 µL per mouse) while positive control animals received 25 mg/kg cyclophosphamide. After 22 h, the animals were injected (i.p.) with colchicine (2 mg/kg) and 2 h later they were sacrificed by cervical dislocation. Bone marrow was expelled from the femur bone with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, cells were fixed three times with meth-anol: acetic acid (3:1). Slides were prepared for bone marrow chromosomes and the slides were stained with giemsa. All the slides were coded and 100 well-spread metaphase cells per animal were scored for CA, i.e., a total of 500 metaphase calls were scored per dose tested. Mitotic index (MI) was scored from 1000 cells/animal and was expressed as percentage. CA was scored following the standard WHO guidelines. The aberration frequencies per cell for chromatid and chromosome types were calculated. Gaps were recorded and not included either as a percentage or as the frequency of aberrations per cell.
Paraffin-coated (approximately 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of mice under diethyl ether anesthe-sia for in vivo SCE study and cell replication kinetic analysis. The test chemical was administered as a single i.p. injection 1 h after the tablet implantation. Three doses (20, 40, and 80 mg/kg) of BESEB CDRI-08 were injected i.p, in distilled water (100 µL/mouse) to different groups of five animals (three males and two females) each. Negative control mice were injected with 100 µL of distilled water while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight. For SCE analysis colchicine (4 mg/kg) was injected (i.p.) 22 h after BrdU tablet implantation. Two hours later, bone marrow was expelled with 0.075 M KC1. After hypotonic treatment (0.075 M KC1 at 37°C) for 20 min cells were fixed three times with methanol: acetic acid (3:1). The slides were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa tech-nique. All slides were coded and 30 s division metaphase cells (40 ± 2 chromo-somes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells (100 cells per animal) were scored for cell replication kinetic analysis by their staining patterns as first (M1), second (M2), and third (M3) division metaphases. The replicative indices (RI) were calculated as follows:
RI = 1M1 + 2M2 + 3M3100No significant increase in the SCE and CA was observed when compared with respective negative controls. No significant changes were observed either in replica-tive index (RI) in SCE study or in the MI in CA study when compared with respec-tive controls. Trend tests for the evidence of dose response effects wave also negative for both SCE and CA studies. The present study of CA and SCE indicates that baco-sides A and B were not genotoxic in bone marrow cells of mice.
The overall results of CA and SCE indicate that the new BESEBB CDRI-08 was not genotoxic in bone marrow cells in vivo in this present experimental condition described earlier.
Ames Salmonella mutagenicity assay has been extensively used to determine the mutagenicity of different drugs. The Salmonella test was first validated in a study of 300 chemicals, most of which are known carcinogens.
In the present study, mutagenicity tests were carried out from BESEB CDRI-08 using tester strains TA97a and TA100 both with and without metabolic activation systems. Inbred Charles River male rats, weighing between 150 and 175 g were used for the preparation of rat liver homogenate (S9) for mutagenicity assays.
Biotin, histidine, dimethyl sulfoxide (DMSO) as surfactant, nicotine adenine dinuccleotide phosphate (NADP), glucose-6-phosphate, crystal violet, ampicil-lin trihydrate, sodium azide, 4-nitro-o-phenylenediamine (NPD), 2-aminofluorene (2-AF), and sodium ammonium phosphate of Sigma Chemical Company (St. Louis, MO) and Agar and beef extract HI Media besides BESEB CDRI-08 were used for the study.
Salmonella tester strains TA97a (showing frame shift mutagenesis) and TA100 (showing base pair mutagenesis) were used for Ames bacterial mutagenecity assay. Before the start of the experiment the genotypes of both the tester strains were confirmed.
The overall results of Ames mutagenicity assay of BESEB CDRI-08 indicate that it was not mutagenic in the aforementioned two strains under the present experimen-tal conditions (Giri and Khan, 1990).
The spurt in preclinical investigation has led to several clinical trials on the various extracts of B. monniera. Here again the investigators from BHU were the first to under-take the clinical trial of the plant. Thus, two single-blind open clinical studies have found memory- and learning-enhancing effects of chronic treatment with alcoholic extract and expressed juice (sweetened with fruit syrup) of B. monniera both in patients with anxiety neurosis (Singh and Singh, 1980) and in children (Sharma et al., 1987).
However, the lead to undertake systematic double-blind, randomized placebo-controlled studies to clinically evaluate the effect of BESEB CDRI-08 on behavioral and cognitive functions in children (6–12 years) suffering from attention deficit hyperactivity disorder (ADHD) and adults (55–70 years) suffering from age associated memory impairment (AAMI) was taken by CDRI.
In the clinical trials on ADHD, only those children diagnosed as cases of ADHD as per the diagnostic criteria of Diagnostic and Statistical Manual of Mental Disorder, 4th edition (DSM-IV), of American Psychiatric Association’s Committee were included. Initially, 88 ADHD children were screened, out of which 40 subjects ful-filled the diagnostic criteria and were included in the study. Twenty children received two 50 mg capsules of BESEB CDRI-08 and 20 children were given two capsules of placebo, identical in shape, size, and color. There were two dropouts from the BESEB CDRI-08 group and seven from the placebo group. The BESEB CDRI-08 was administered for 12 weeks daily and from 13th week to 16th week all the chil-dren were given placebo only. They were evaluated initially on day 0, and there-after at 4, 8, 12 weeks of drug administration. The last evaluation was done after 4 weeks of stopping the medication when all children were given placebo only. The tests applied were personal information, mental control, sentence repetition, logi-cal memory, word recall (both meaningful and non-meaningful words), digit span, picture recall, delayed response, and paired associate learning. Significant to highly significant results were obtained in all the parameters after 4–8 weeks.
These clinical trials were carried out at two centers, viz., BRD Medical College, DDU Gorakhpur University, Gorakhpur in 2000 (Sharma, 2000) and KG Medical College, University of Lucknow in 2001 (Raghav et al., 2006; Singh and Sangeetha, 2001).
Subjects with complaints of memory loss in everyday life, e.g., difficulty in remembering names, misplacing objects, difficulty in remembering works which should be done, etc., were recruited by announcement in radio programs, advertise-ments in local news papers, and distribution of hand bills, etc. The inclusion criteria were the following: subjects should be 55 years and older, should have logical mem-ory subtest of Wechsler’s memory scale with a cut off score of =6, a score of 24 or higher on Mini Mental Scale Examination (MMSE) to exclude dementia. Evidence of delirium, confusion, neurological disorder, psychiatric disorder, malabsorption disorder, history of alcoholism, drug abuse, medication known to influence CNS, history of serious disorders of heart, kidney, or bone marrow, etc., were excluded from the study.
Thus, 20 cases in BESEB CDRI-08 and also in placebo groups were each taken at both the centers. The test tools administered were Wechsler Memory Scale— Revised (Wechsler, 1987), MMSE (Folstein et al., 1975), and dosage record treat-ment emergent symptom scales (DOTES, 1985). In the study, the Hindu version of some parts of Wechsler Memory Scale were also used (Chandra, 1980). The fol-lowing parameters were used in the Wechsler Memory Scale: personal and current, orientation, mental control, passages, digits forward, digits backward, visual repro-duction, associated learning, total raw score, correct score, and mental quotient.
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