- Email: [email protected]
Ayurvedic processed seeds of nux-vomica: Neuropharmacological and chemical evaluation
Fitoterapia 81 (2010) 190–195
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Ayurvedic processed seeds of nux-vomica: Neuropharmacological and chemical evaluation☆ Chandrakant Katiyar a,⁎,1, Abhishek Kumar b, S.K. Bhattacharya c,†, R.S. Singh d,† a b c d
323/S, 1st Floor, Southend Floors, Sector 48-49, Sohna Road, Gurgaon (HR), India Department of Pharmaceutical Sciences, Birla Institute of Technology, MESRA, Ranchi, India Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Department of Rasashashtra and Bhaisajya kalpana, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
a r t i c l e
i n f o
Article history: Received 2 March 2009 Accepted in revised form 13 August 2009 Available online 21 August 2009 Keywords: Ayurvedic processings CNS stimulant Detoxification Neuropharmacology Strychnos nux-vomica Shodhan
a b s t r a c t The effect of detoxification on Strychnos nux-vomica seeds by traditional processing with aloe and ginger juices (B), by frying in cow ghee (C), and by boiling in cow milk (D) was investigated. The ethanolic extracts of these samples were subjected to spontaneous motor activity (SMA), pentobarbitone-induced hypnosis, PTZ induced convulsions, diazepam-assisted protection, and morphine-induced catalepsy. All samples reduced SMA and inhibited catalepsy. The seeds processed in milk (D) showed the lowest strychnine content in the cotyledons, exhibited marked inhibition of PTZ induced convulsions and maximal potentiation of hypnosis, and were the safest (LD50). © 2009 Elsevier B.V. All rights reserved.
1. Introduction Strychnos nux-vomica (Linn.) belonging to Fam: Loganiaceae has been a very promising drug for certain disorders. As such, nux-vomica is mostly used in disorders of the gastrohepatic tract. It is an important remedy for atony and relaxation of the stomach and bowels and disorders dependent thereon. Apart from its gastro-hepatic uses, the plant in Ayurvedic classics [1] has been reported to be used as analgesic, stimulant, also useful in impotence, spermatorrhea, and sexual frigidity of women. It is said to be useful in the
☆ Submitted at the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. This paper was presented at 59th Indian Pharmaceutical Congress. ⁎ Corresponding author. Research and Development (Ayurveda), R&D Centre, Dabur India Ltd., Sahibabad, India. Tel.: +91 124 4253323; fax: +91 124 2343545. E-mail addresses: [email protected], [email protected] (C. Katiyar). 1 Part of M.D.(Ayu.) Thesis, 1983. † Deceased. 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.023
urinary incontinence of children and elderly, when it is due to a relaxed or paralyzed sphincter. It is used as a nervine tonic and aphrodisiac [2–4]. It is included as a constituent in various traditional Ayurvedic formulations such as Agnitundi Rasa, Navjivan Rasa, Shoolnirmoolan Rasa etc. [5]. Not less than sixty formulations have been reported in the literature of the Indian systems of medicine (ISM) which include nuxvomica, of which approximately thirty formulations are used in the disorders of vāta dosha (mainly responsible for neurological disorders and pain). Pain, as per the philosophy of Ayurveda, is attributed to vata dosha, among the three doshas i.e. vata, pitta and kapha. Ancient Ayurvedic texts suggest use of any toxic drug after suitable processing or detoxification, to reduce or modify the effects/poisonous effects of the drug and render it suitable for use in formulations for therapeutic use. The processings are said to bring about favorable changes that modifies the therapeutic effect and also renders the drug free from poisonous effects. Being a poisonous drug this drug (nuxvomica) has also been used after shodhan (detoxification), for therapeutic purposes [5]. Different methods have been quoted in the classical texts of Ayurveda for a single drug
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
[1,5–10]. Methods that are often practiced and are recommended in the texts of Ayurveda for S. nux-vomica were considered for the study with an aim to provide a comparative profile and generate statistical data behind use of the method(s). The manuscript discusses the qualitative and quantitative pharmacological change in the seeds after processing. The processings were performed in exactly the similar conditions as is practiced by the practitioners, so that their mutual qualitative relativity can be better understood. The pharmacological studies were planned, considering the reported use of nux-vomica in neurological disorders in the Indian system of medicine.
191
Sample C: The seeds were washed, dried and fried in cow ghee (clarified butter) till brownish red. The swollen seeds were then powdered [10,11]. Sample D: The seeds were tied in a cotton cloth of fine mesh size. The cloth containing the seeds was hanged in a vessel containing boiling cow milk, (q. s. to properly immerse the seeds) for 3 h. The milk was added intermittently to maintain the volume of the milk. The seeds after 3 h were washed with potable water, peeled and the embryo removed. The cotyledons so obtained were then powdered [12]. 2.4. Extraction and preparation of the stock solution for pharmacological screening
2. Materials and methods 2.1. Animals Swiss albino mice (20–25 g) and Wistar rats (150–200 g) of either sex from the animal house of Institute of Medical Sciences, Banaras Hindu University, Varanasi were used for the experiment. The animals were housed in groups of five each and were housed according to standard laboratory conditions of temperature (25 ± 1 °C), relative humidity (55 ± 5%) and lighting (07:00–19:00 h). All animals were fasted overnight before test and water was supplied ad libitum. The ambient temperature was 25 ± 1 °C. Behavioral observations took place in the forenoon and each animal was used only once. Animal ethical committee approval and animal house registration number could not be added since the work was carried out in 1983.
Processed and unprocessed nux-vomica were ground to coarse powder, passed through sieve no. 40. The powder (150 g) was packed in a soxhlet extractor. The drug was extracted with 95% alcohol till exhaustion of the active principle. The extract thus obtained was concentrated in vacuo to 50 ml (1 ml ≈ 3 g of drug). 5 ml of extract of test drug was evaporated to dryness. The residue was made into a suspension using two drops of Tween 80. The volume was adjusted to 60 ml. 1 ml of this solution was diluted to 25 ml with distilled water (stock solution). The stock solution was further diluted ten times to get the test dose suspension. The pharmacological effect of the test sample (suspension) was observed at the sub-convulsive dose level. The extract was administered intraperitonially to the animals. 2.5. Pharmacological studies
2.2. Chemical Pentobarbitone, morphine, pentylenetetrazol (Cardiazol), diazepam, ammonia, sulphuric acid and chloroform. Nuxvomica (voucher specimen number: CKK/IMS/RSBK/81–82) was purchased from a local supplier of Banaras. The processing materials like cow ghee and self extracted juices were freshly prepared. Fresh cow milk was procured from a local supplier.
2.5.1. Acute toxicity test 150 Swiss albino mice were divided into groups having five animals each. The test samples were administered in graded doses by i.p. injection to the animals. The animals were kept under continuous observation for 24 h and any mortality was checked. The animals were further observed for the next 14 days. Log dose–probit mortality curves were drawn in accordance to Karber's method and the median lethal dose [13].
2.3. Ayurvedic processings (Shodhan) The seeds of nux-vomica from a single batch were divided into four lots and subjected to various methods of detoxification. Sample A: The crude nux-vomica seeds were steamed to facilitate the removal of seed coat and embryo. The cotyledons so obtained were then used in the study as reference to compare the changes that occur after processings. Sample B: The seeds were kept immersed in the exudate scraped from the fresh leaves and stems of Aloe vera (ghritakumari) covered with a lid and kept aside for 15 days, with occasional stirring. On the last day the seeds were washed. The swollen seeds were again immersed in the juice of Zingiber officinale (ardraka) and kept in a cool place for 7 days. The seed coat and the embryo were removed from the seeds. The cotyledons were finally powdered. This method was adopted on the basis of a survey of few Ayurvedic industries, which were preparing shuddha (processed) nux-vomica for use in different formulations, though it was not mentioned in the textbooks.
2.5.2. Pentobarbital-sleeping time Twenty-five mice (20–25 g) were divided into five groups of five mice each. In four out of five groups, the test samples were administered and the fifth group served as control. All animals received sodium pentobarbital (50 mg/kg i.p.) 30 min later. The loss of the righting reflex, after the administration of pentobarbital was recorded as onset of sleep, while the time from the loss to regaining of the righting reflex, as the duration of sleep. Increase in the sleeping time over control was taken as the potentiation of the pentobarbitone hypnosis [14]. 2.5.3. Spontaneous motor activity (SMA) The tunnel board was used to record the spontaneous motor activity. The tunnel board measured 61 × 61 cm constituted twelve tunnels (7.5 cm) and 4 cm in diameter, arranged in a symmetrical manner. Mice were placed one at a time on the right hand corner of the tunnel board. Test samples were administered i.p. at sub-convulsive doses and
192
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
the activity was recorded after 30, 45, and 60 min. The number of the tunnel crossed and the total number of tunnels crossed were recorded [15]. 2.5.4. Pentylenetetrazole (PTZ) induced convulsion test Swiss albino mice were divided into five groups (n = 5) and treated with the test samples or saline. PTZ was administered (80 mg/kg s.c.) after 15 min of administration of test samples. Animals were then observed (60 min) for occurrence of seizures and the presence or absence of clonic convulsions. The duration between administration of PTZ and onset of convulsion and mortality, was recorded. An episode of convulsions persisting for a period of at least 10 s was considered as threshold convulsion. Animals devoid of seizures were considered protected [16]. 2.5.5. Effect of diazepam on lethality of crude and processed nux-vomica in mice Diazepam (5 mg/kg, i.p.) was injected into groups of mice. 30 min later the LD100 dose of test samples was injected i.p. The mice were observed for the presence of convulsions, nature of convulsions and mortality if any, for 24 h. 2.5.6. Morphine-induced catalepsy in rats Twenty-five Wistar rats were divided in five groups. Group 1 served as control and the rest of the groups were treated with the test samples in order of samples I, II, III, and IV. After half an hour of the treatment with the test sample the animals were administered with morphine hydrochloride (10 mg/kg i.p.). Catalepsy was measured as immobility percentage. Four distinct stages were discernible; Stage I: the animal sat quietly, with no attempt to move, unless pushed gently, Stage II: the animal didn't move even on being pushed, Stage III: the forepaws were gently put on lower retort ring (7.5 cm high) of the catalepsy stand. The animal maintained the posture for 30 s or more, Stage IV: by raising one of the hind limbs and then removing the support, the raised limb continues to remain suspended in the air. Preservation of this posture for 30 s or more was considered the prerequisite for the onset of stage IV of the catalepsy. Once the animal reached the stage IV or the deepest possible stage, it was subjected to the ring test. The cataleptic animal was gently lifted by its tail and placed on the upper ring (40 cm high) of the cataleptic stand in such a manner that only the limbs of the animal rested on the ring. Once placed, the animal was closely observed for a period of 5 min. The total period for which the animal remained perfectly immobile was taken to represent the depth of catalepsy as “immobility percentage”. 2.6. Phytochemical study 2.6.1. Extraction and quantification The total alkaloidal content was determined by a modified B.P. 1980 method. About 0.4 g accurately weighed fine powder of the drug was saturated with dil. ammonia solution and extracted with 60 ml chloroform in a soxhlet apparatus of suitable size for 2 h. The chloroform extract was then treated with four quantities of sulphuric acid (0.1 N; 20 ml). The combined acid solution was washed with chlo-
roform (20 ml). The aqueous solution after filtration was warmed gently over the water bath in a shallow dish, to remove the traces of chloroform. The solution was cooled and diluted to 100 ml with sulphuric acid (0.1 N). 20 ml of this solution was again diluted to 100 ml with 0.1 N sulphuric acid. The extinction of 1 cm layer was measured at 262 nm and 330 nm [17]. 2.6.2. Assay of strychnine in powdered drug The processed seeds were coarsely powdered. They were then boiled in hydrochloric acid (50%; 20 ml) and filtered. After filtration, the aqueous portion was extracted with four quantities of chloroform (25 ml). The aqueous portion was made alkaline with the dilute ammonia solution drop wise and again extracted with four quantities of chloroform (25 ml). The chloroform extract was then combined and evaporated to dryness. The residue so obtained was dissolved in a mixture of sulphuric acid (3%; 15 ml) and nitric acid (2 ml). Few crystals of sodium nitrite were added to the solution. After about 40 min at room temperature, the solution was again made alkaline with 10% sodium hydroxide solution and the alkaline solution was then extracted with chloroform (20 ml). The chloroform layer was washed with sodium hydroxide (5 ml) and then with two quantities of water (10 ml). Each chloroform extract thus processed similarly. The second aqueous washing was titrated with sulphuric acid (0.1 N) using methyl red as indicator. If more than 0.1 ml was required, the chloroform solution was washed further with water until the aqueous washing required not more than 0.1 ml of 0.1 N sulphuric acid for neutralization. The chloroform solution was then dried with the help of anhydrous sodium sulphate and filtered. It was then evaporated to dryness. The residue was dissolved in sulphuric acid (0.1 N; 10 ml) and an excess of acid back titrated with sodium hydroxide (0.1 N). Each ml of sulphuric acid (0.1 N) is equal to 0.03344 g of strychnine. The result was multiplied with 1.02 to correct for the loss of strychinine [18]. 2.6.3. Chromatographic analysis All the samples were chromatographed as crude alkaloid fraction (CAF) on silica gel TLC plate with solvent system (chloroform:methanol; 95:5 v/v) and spray of Dragendorff's reagent was used for detection. All the test drug samples were analyzed for the total alkaloidal content in the different portions of the seeds and total contents of strychnine. 2.7. Statistical analysis The values are expressed as mean ± SEM. Statistical significance was analyzed by ANOVA followed by Dunnet's T test. P < 0.05 was considered to be significant. 3. Results 3.1. Pharmacological studies 3.1.1. LD50 dose determination Nux-vomica extract, when administered i.p. to the animals, showed a great variation in the toxic dose. The results have been summarized in the Table 1.
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195 Table 1 LD50 values (method of Karber) of intraperitonially administered extract and percentage of strychnine in differently processed samples of nux-vomica seed.
Table 4 Effect of crude and processed nux-vomica on PTZ induced convulsions in mice. Groups
S. no.
1 2 3 4
Sample
Crude drug (sample A) Aloe and ginger treated (sample B) Ghrit treated (sample C) Milk treated (sample D)
LD50 value (mg/kg)
Percentage of strychnine (% w/w) Test drug
Seed coat
52
2.1875
0.4336
70
1.29
0.745
205
1.212
0.0525
600
0.4375
1.12
3.1.2. Effect of crude and processed nux-vomica on pentobarbitone-induced hypnosis in mice The mean sleeping time of animals treated with pentobarbital sodium (50 mg/kg i.p.) was 29.8 ± 2.87 mins. Pretreatment of the animals at sub-convulsive doses with the crude and treated samples of nux-vomica enhanced pentobarbitone-induced sleeping time. Milk treated sample showed the maximal potentiation followed by samples C, A and B. Intermittent convulsions were noted in the crude drug sample group (sample A) even while the mice were asleep. Such convulsions were also present in some mice, treated with sample B but were absent in those treated with sample C and D (Table 2). 3.1.3. Effect of crude and processed nux-vomica on spontaneous motor activity in mice The effect of the test drugs on Shillito's tunnel board shows that the crude nux-vomica (sample A) markedly Table 2 Effect of crude and processed nux-vomica at sub-convulsive doses on pentobarbitone-induced hypnosis in mice. S. no
Dose (mg/kg)
Test sample
Sleeping time (min)
1 2 3 4 5
10 ml/kg 20 25 70 125
Control Sample A Sample B Sample C Sample D
29.8 ± 2.87 47.6 ± 3.18⁎⁎ 35.6 ± 6.60⁎ 48.0 ± 3.91⁎⁎⁎ 53.8 ± 2.76⁎⁎⁎
Values are expressed as mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎⁎P < 0.01, ⁎P > 0.05, ⁎⁎⁎P < 0.0001. ANOVA followed by Dunnett's T test signifies the test.
193
Control Sample A Sample B Sample C Sample D
Dose (mg/kg)
Time interval between administration of drug and onset of convulsion (min)
20 25 70 125
6.0 ± 0.92 4.75 ± 1.03⁎ 8.0 ± 0.82⁎ 10.0 ± 0.41⁎⁎ 11.25 ± 0.48⁎⁎⁎
Values are expressed as Mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎P > 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.0001. ANOVA followed by Dunnett's T test signifies the test.
reduced the number of tunnels entered at 30, 45 and 60 min of observation time. A similar reduction was noted with samples C and D at similar observation time. There wasn't a significant difference between the results obtained with mice of C and D group throughout the experiment, though the numbers of tunnel entries were slightly more than that of the crude sample group at 45 and 60 min. There was a gradual decrease in the number of entries with sample A treated group. All the treatments were at sub-convulsive doses (Table 3). 3.1.4. Effect of crude and processed nux-vomica on PTZ induced convulsions in mice The effects of the processed and unprocessed samples were compared with pentylenetetrazole (80 mg/kg i.p.) produced convulsion in the experimental animals. The mean time for the onset of convulsions was 6 min. PTZ induced convulsions were markedly inhibited by sample D significantly and to an insignificant extent by sample C. Crude nux-vomica reduced the time of onset of convulsions. The maximum time for onset was noted with sample D followed by C and B (Table 4). 3.1.5. Effect of diazepam on lethality of crude and processed nux-vomica in mice The effect of administration of diazepam 30 min before the test samples was observed to cause any protection against the 100% lethal dose (LD100) of nux-vomica samples. The LD50 values were calculated previously with the method of Karber. Diazepam completely inhibited the lethal effect of samples A, C and D at their respective LD100 values. There was an incomplete but significant inhibition of mortality induced by sample B. Samples A and B produced intermittent convulsions predominantly tonic in nature, while convulsions of clonic
Table 3 Effect of crude and processed nux-vomica on spontaneous motor activity in mice. Time interval between administration of drug and observation (min)
N
Mean number of tunnels entered A
B
C
D
0 30 45 60
5 5 5 5
25.2 ± 0.8 11.0 ± 0.8944⁎ 8.0 ± 0.4472⁎ 4.8 ± 0.5831⁎
25.2 ± 0.9165 29.4 ± 1.030 13.4 ± 0.6782⁎ 13.2 ± 0.5831⁎
20.8 ± 0.8602 4.0 ± 0.6325⁎ 10.8 ± 0.6633⁎ 10.4 ± 0.7483⁎
21.4 ± 0.9274 6.0 ± 0.4472⁎ 13.2 ± 0.8602⁎ 15.0 ± 0.7071⁎
Values are expressed as mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎P < 0.01, ANOVA followed by Dunnett's T test signifies the test.
194
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
and tonic nature were produced by sample C. Sample D produced convulsions predominantly clonic in nature, followed by few episodes of tonic convulsions. 3.1.6. Morphine-induced catalepsy in rats Morphine (10 mg/kg i.p.) produced marked cataleptic effect in rats. Most of the animals passed phase III of the catalepsy with a mean cataleptic score (MCS) of 77.2. Pretreatment with crude nux-vomica produced a slight but significant potentiation of morphine-induced catalepsy. The treatment with samples C and D antagonized the morphineinduced catalepsy (Table 5). 3.2. Phytochemical studies The different components of the seeds after processing were extracted and the amount of total alkaloids was determined. The results have been compiled in Table 6. The extract obtained from the cotyledons of the differently processed nux-vomica was also chromatographed using chloroform:methanol (95:5 v/v) on a silica gel plate and Dragendorff's reagent as spotting agent. The chromatogram showed the presence of a new spot in the nux-vomica processed with milk. Further isolations and characterizations of the new spot shall be discussed elsewhere. The total strychnine contents were determined using modified B.P. 1980 method (Tables 1 and 6). 4. Discussion In the present study, an attempt was made to analyze the pharmacological efficacy of S. nux-vomica, before and after processing (shodhan) it with the methods quoted in
Table 5 Effect of crude and processed nux-vomica on morphine-induced catalepsy in mice. S. no.
Treatment groups
Immobility (%) Mean ± S.E.
Percentage difference with regard to control group
1 2 3 4 5
Control Sample A Sample B Sample C Sample D
77.20 ± 3.79 85.40 ± 1.77⁎ 0±0 0±0 24.20 ± 10.11⁎⁎
– 10 (+) 100 (−) 100 (−) 68.65 (−)
(+) indicates potentiation; (−) indicates inhibition in the effect.
Table 6 Percentage of total alkaloids in different portions of seeds of processed drug samples. S. no.
Drug
1
Aloe and ginger treated (sample B) Ghrit treated (sample C) Milk treated (sample D)
2 3
Percentage of total alkaloids (% w/w) Cotyledon
Seed coat
Embryo
80.6
15.3
0.2
75
13.3
–
71.6
12.5
0.2
the classical Ayurvedic literature, so that their mutual qualitative relativity can be better understood. Shodhan is one of the important processes described in the ancient literatures of Ayurveda with reference to the use of toxic substances including metals, minerals and plants. It is presumed that the technique was advised and used by the ancient scholars to modify/enhance the pharmacological property and reduce or diminish the poisonous effects of any toxic drug and render it suitable to be used therapeutically. Nux-vomica is rarely used in practice as such, due to high strychnine content being opisthotonous in the modern system of medicine, although it is widely used in the alternative system of medicines even today, since they have their own ways of formulating and handling the toxic drugs. The drug is found to be useful in the urinary incontinence of children and elderly when it is due to a relaxed or paralyzed sphincter. It stimulates the sexual organs and hence given with varying success in impotence, spermatorrhea, and sexual frigidity of women. Nux-vomica is one of the best agents for so-called chronic G.I. ailments of various types and origins, but all of an atonic character. It is an important remedy for atony and relaxation of the stomach and bowels and disorders dependent thereon. The comparative neuropharmacological studies were planned keeping in view the clinical use of treated nuxvomica seeds in Ayurveda. Processing of the seeds effectively affected the toxic dose as exhibited in the Table 1. The difference in toxic doses may be attributed to change in total alkaloidal contents and strychnine. Nux-vomica showed significant pentobarbitoneinduced hypnosis potentiating action. However, animals of the crude nux-vomica group (sample A) showed convulsions during sleep that were found to be absent in the animals treated with processed nux-vomica. In addition, the observations indicate that the processings apart from reducing the toxicity, potentiated the barbiturate induced hypnosis. It was also seen that the seeds treated with milk (sample D) contains less amount of strychnine as compared to others, but has a much better response than other samples. This is indicative of the fact that nux-vomica after processing tends to exhibit a CNS depressant action, at sub-convulsive doses. All the samples were found to reduce the locomotor activity of the animals in the Shillito's tunnel board test. Since, exploratory behavior is an inherent attribute of this experimental model, the results indicate that in sub-convulsive doses, crude as well as processed (shodhit) nux-vomica inhibited the exploratory behavior which is again indicative of CNS depressive type action. PTZ is a convulsant known to act on the reticular activating system of brain stem and also on motor cortex. PTZ convulsions are used to evaluate anti-epileptic drugs likely to be used in petitmal epilepsy. Crude nux-vomica (sample A) hastened the onset of PTZ convulsions as was expected, both of them (PTZ and nux-vomica) being established convulsive agents. On the contrary, all the processed samples increased the time of onset of convulsions, and the maximum delay in onset was seen with sample D where the convulsions were inhibited to a significant duration. The results indicate that processing the seeds with milk causes the reversal of the convulsive effect of nux-vomica. Processing with milk imparts the maximal anticonvulsant effect. This
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
action may be attributed to the lesser strychnine content and other chemical transformations that would have occurred during the processing. Catalepsy is known to be associated with rigidity of skeletal muscles brought about by increased motor tone, subsequent to striatal dysfunction. The fact that processed samples exert an anticataleptic action (Table 5) can justify the use of nux-vomica in clinical condition of Parkinson's disease in which muscular rigidity is a sign. Diazepam is clinically used as an antidote in strychnine poisoning. The effect of diazepam (5 mg/kg i.p.) was observed on the mortality induced by crude and processed samples when administered 30 min before the administration of test drug samples. The doses of all the test samples chosen were the 100% lethal doses (LD100). Diazepam completely inhibited the lethal effects of samples A, C and D. There was an incomplete but significant inhibition of mortality induced by sample B (200 mg/kg i.p.). Crude nux-vomica (sample A) and sample B produced intermittent convulsions which were predominantly tonic in nature. Animals treated with sample C also exhibited intermittent convulsions which were clonic and tonic in nature, whereas with sample D the convulsions were predominantly clonic, followed by episodes of tonic convulsions. Experimental catalepsy is regarded as a laboratory counterpart of clinical Parkinsonism. Morphine catalepsy has been investigated and found to be mediated through striatal cholinergic mechanisms [19,20]. The unprocessed nux-vomica extract (sample A) produced a potentiating effect whereas the processed samples inhibited morphine-induced catalepsy (Table 5). The quantitative studies on alkaloids suggest that the content of alkaloids decreases in order from sample A > B > C > D. The order is similar for strychnine content also (Tables 1 and 6). In the morphine-induced catalepsy, the results show that treatment with sample A produces slight potentiation of the morphine-induced catalepsy, while samples B and C completely antagonized this effect and the sample D which earlier exhibited the maximum potentiation in the hypnosis potentiation could not reduce the morphine-induced catalepsy to a significant extent. These studies suggest that the individual cases of treatment cannot be correlated merely on the basis of strychnine contents and neither can it be assumed to be due to spinal motor neuron stimulatory effect of the alkaloid. From these experiments, it was inferred that nux-vomica after processings tends to suppress spontaneous movements, reduces initiative and interest in environment and there was some potentiation of hypnosis. Therefore, it may be concluded that the shodhan treatment brings about some phyto-
195
chemical changes that in turn reverses the pharmacological profile of the drug. The present investigation for the first time, reports that that there is a weak to moderate degree of CNS depressant activity that justifies the use of the processed drug clinically in various neurological disorders like Parkinson's disease and Trigeminal neuralgia for which satisfactory treatment is still awaited. This study also provides an indication of which method to use to attain the desired efficacy and results among those quoted in the texts. However, these pharmacological changes justify the need of detailed phytochemical investigations with reference to changes in phytochemical composition responsible for transformed biological activity. Acknowledgements CK thankfully acknowledges the Dean, Faculty of Ayurveda Head, Department of Rasashashtra and Head, Department of Pharmacology, IMS, BHU for providing the necessary facilities, help and guidance during the tenure. References [1] Bhavamisra. Bhavprakash. Varanasi. Chaukhambha Sanskrit Sansthan; 2002. p. 568. [2] Shashtri MU. Prayogatmak Abhinav Dravyaguna Vigyanam. Patna: Baidyanath Ayurved Bhawan; 1991. p. 266. [3] Gaud SD. Shankar Nighantu. Jabalpur: Banaushadhi Bhandar 1935:96. [4] Singh RS. Vanaushadhi Nirdishika. Lucknow: Uttar Pradesh Hindi Sansthan 1983:1089. [5] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas 1979:679. [6] Anonymous. The Ayurvedic formulary of India (AFI), part I. Ministry of Health and Family Planning, Dept. of Health, Govt. of India, Delhi; 1978. p. 362. [7] Sharma R. Arogya Prakash. Patna: Baidyanath Ayurved Bhawan, 21st ed.; 1982. p. 87. [8] Vaidya B. Nighantu Adarsh. Varanasi: Chaukhambha Sanskrit Sansthan, 2nd ed.; 1978. p. 62. [9] Krishnanandji. Rasatantra Sāra Evam Siddha Prayoga Sangraha. Kaleda, Ajmer: Krishnagopal Ayurved Bhavan; Part I, 8th Edition; 1999. p. 75. [10] Shashtri L. Yogaratnakar. Varanasi: Chaukhambha Sanskrit Sansthan, 7th ed.; 1999. p. 168. [11] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas; 1979. p. 174–5. [12] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas; 1979. p. 176–7. [13] Ghosh MN. Fundamentals of experimental pharmacology. Kolkatta: Bose Printing House; 2005. p. 197. [14] Ramirez TED, Ruriz NN, Arellano JDQ, Madrigal BR, Michel MTV, Garzon P. J Ethnopharmacol 1998;61:143–52. [15] Shillito EE. Br J Pharmacol 1970;40:113. [16] Adzu B, Amos S, Muazzam I, Inyang US, Gamaniel KS. J Ethnopharmacol 2002;83:139–43. [17] Anonymous. British Pharmacopoeia- II. Cambridge: University Press; 1980. p. 564. [18] Anonymous. British Pharmacopoeia- II. London: General Medical Council Pharmaceutical Press; 1958. p. 510. [19] Bose R, Bhattacharya SK. Indian J Med Res 1979;70:281–8. [20] Bose R, Kumar A, Bhattacharya SK. Neurosci Lett 1979;14:115–8.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Ayurvedic processed seeds of nux-vomica: Neuropharmacological and chemical evaluation☆ Chandrakant Katiyar a,⁎,1, Abhishek Kumar b, S.K. Bhattacharya c,†, R.S. Singh d,† a b c d
323/S, 1st Floor, Southend Floors, Sector 48-49, Sohna Road, Gurgaon (HR), India Department of Pharmaceutical Sciences, Birla Institute of Technology, MESRA, Ranchi, India Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Department of Rasashashtra and Bhaisajya kalpana, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
a r t i c l e
i n f o
Article history: Received 2 March 2009 Accepted in revised form 13 August 2009 Available online 21 August 2009 Keywords: Ayurvedic processings CNS stimulant Detoxification Neuropharmacology Strychnos nux-vomica Shodhan
a b s t r a c t The effect of detoxification on Strychnos nux-vomica seeds by traditional processing with aloe and ginger juices (B), by frying in cow ghee (C), and by boiling in cow milk (D) was investigated. The ethanolic extracts of these samples were subjected to spontaneous motor activity (SMA), pentobarbitone-induced hypnosis, PTZ induced convulsions, diazepam-assisted protection, and morphine-induced catalepsy. All samples reduced SMA and inhibited catalepsy. The seeds processed in milk (D) showed the lowest strychnine content in the cotyledons, exhibited marked inhibition of PTZ induced convulsions and maximal potentiation of hypnosis, and were the safest (LD50). © 2009 Elsevier B.V. All rights reserved.
1. Introduction Strychnos nux-vomica (Linn.) belonging to Fam: Loganiaceae has been a very promising drug for certain disorders. As such, nux-vomica is mostly used in disorders of the gastrohepatic tract. It is an important remedy for atony and relaxation of the stomach and bowels and disorders dependent thereon. Apart from its gastro-hepatic uses, the plant in Ayurvedic classics [1] has been reported to be used as analgesic, stimulant, also useful in impotence, spermatorrhea, and sexual frigidity of women. It is said to be useful in the
☆ Submitted at the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. This paper was presented at 59th Indian Pharmaceutical Congress. ⁎ Corresponding author. Research and Development (Ayurveda), R&D Centre, Dabur India Ltd., Sahibabad, India. Tel.: +91 124 4253323; fax: +91 124 2343545. E-mail addresses: [email protected], [email protected] (C. Katiyar). 1 Part of M.D.(Ayu.) Thesis, 1983. † Deceased. 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.023
urinary incontinence of children and elderly, when it is due to a relaxed or paralyzed sphincter. It is used as a nervine tonic and aphrodisiac [2–4]. It is included as a constituent in various traditional Ayurvedic formulations such as Agnitundi Rasa, Navjivan Rasa, Shoolnirmoolan Rasa etc. [5]. Not less than sixty formulations have been reported in the literature of the Indian systems of medicine (ISM) which include nuxvomica, of which approximately thirty formulations are used in the disorders of vāta dosha (mainly responsible for neurological disorders and pain). Pain, as per the philosophy of Ayurveda, is attributed to vata dosha, among the three doshas i.e. vata, pitta and kapha. Ancient Ayurvedic texts suggest use of any toxic drug after suitable processing or detoxification, to reduce or modify the effects/poisonous effects of the drug and render it suitable for use in formulations for therapeutic use. The processings are said to bring about favorable changes that modifies the therapeutic effect and also renders the drug free from poisonous effects. Being a poisonous drug this drug (nuxvomica) has also been used after shodhan (detoxification), for therapeutic purposes [5]. Different methods have been quoted in the classical texts of Ayurveda for a single drug
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
[1,5–10]. Methods that are often practiced and are recommended in the texts of Ayurveda for S. nux-vomica were considered for the study with an aim to provide a comparative profile and generate statistical data behind use of the method(s). The manuscript discusses the qualitative and quantitative pharmacological change in the seeds after processing. The processings were performed in exactly the similar conditions as is practiced by the practitioners, so that their mutual qualitative relativity can be better understood. The pharmacological studies were planned, considering the reported use of nux-vomica in neurological disorders in the Indian system of medicine.
191
Sample C: The seeds were washed, dried and fried in cow ghee (clarified butter) till brownish red. The swollen seeds were then powdered [10,11]. Sample D: The seeds were tied in a cotton cloth of fine mesh size. The cloth containing the seeds was hanged in a vessel containing boiling cow milk, (q. s. to properly immerse the seeds) for 3 h. The milk was added intermittently to maintain the volume of the milk. The seeds after 3 h were washed with potable water, peeled and the embryo removed. The cotyledons so obtained were then powdered [12]. 2.4. Extraction and preparation of the stock solution for pharmacological screening
2. Materials and methods 2.1. Animals Swiss albino mice (20–25 g) and Wistar rats (150–200 g) of either sex from the animal house of Institute of Medical Sciences, Banaras Hindu University, Varanasi were used for the experiment. The animals were housed in groups of five each and were housed according to standard laboratory conditions of temperature (25 ± 1 °C), relative humidity (55 ± 5%) and lighting (07:00–19:00 h). All animals were fasted overnight before test and water was supplied ad libitum. The ambient temperature was 25 ± 1 °C. Behavioral observations took place in the forenoon and each animal was used only once. Animal ethical committee approval and animal house registration number could not be added since the work was carried out in 1983.
Processed and unprocessed nux-vomica were ground to coarse powder, passed through sieve no. 40. The powder (150 g) was packed in a soxhlet extractor. The drug was extracted with 95% alcohol till exhaustion of the active principle. The extract thus obtained was concentrated in vacuo to 50 ml (1 ml ≈ 3 g of drug). 5 ml of extract of test drug was evaporated to dryness. The residue was made into a suspension using two drops of Tween 80. The volume was adjusted to 60 ml. 1 ml of this solution was diluted to 25 ml with distilled water (stock solution). The stock solution was further diluted ten times to get the test dose suspension. The pharmacological effect of the test sample (suspension) was observed at the sub-convulsive dose level. The extract was administered intraperitonially to the animals. 2.5. Pharmacological studies
2.2. Chemical Pentobarbitone, morphine, pentylenetetrazol (Cardiazol), diazepam, ammonia, sulphuric acid and chloroform. Nuxvomica (voucher specimen number: CKK/IMS/RSBK/81–82) was purchased from a local supplier of Banaras. The processing materials like cow ghee and self extracted juices were freshly prepared. Fresh cow milk was procured from a local supplier.
2.5.1. Acute toxicity test 150 Swiss albino mice were divided into groups having five animals each. The test samples were administered in graded doses by i.p. injection to the animals. The animals were kept under continuous observation for 24 h and any mortality was checked. The animals were further observed for the next 14 days. Log dose–probit mortality curves were drawn in accordance to Karber's method and the median lethal dose [13].
2.3. Ayurvedic processings (Shodhan) The seeds of nux-vomica from a single batch were divided into four lots and subjected to various methods of detoxification. Sample A: The crude nux-vomica seeds were steamed to facilitate the removal of seed coat and embryo. The cotyledons so obtained were then used in the study as reference to compare the changes that occur after processings. Sample B: The seeds were kept immersed in the exudate scraped from the fresh leaves and stems of Aloe vera (ghritakumari) covered with a lid and kept aside for 15 days, with occasional stirring. On the last day the seeds were washed. The swollen seeds were again immersed in the juice of Zingiber officinale (ardraka) and kept in a cool place for 7 days. The seed coat and the embryo were removed from the seeds. The cotyledons were finally powdered. This method was adopted on the basis of a survey of few Ayurvedic industries, which were preparing shuddha (processed) nux-vomica for use in different formulations, though it was not mentioned in the textbooks.
2.5.2. Pentobarbital-sleeping time Twenty-five mice (20–25 g) were divided into five groups of five mice each. In four out of five groups, the test samples were administered and the fifth group served as control. All animals received sodium pentobarbital (50 mg/kg i.p.) 30 min later. The loss of the righting reflex, after the administration of pentobarbital was recorded as onset of sleep, while the time from the loss to regaining of the righting reflex, as the duration of sleep. Increase in the sleeping time over control was taken as the potentiation of the pentobarbitone hypnosis [14]. 2.5.3. Spontaneous motor activity (SMA) The tunnel board was used to record the spontaneous motor activity. The tunnel board measured 61 × 61 cm constituted twelve tunnels (7.5 cm) and 4 cm in diameter, arranged in a symmetrical manner. Mice were placed one at a time on the right hand corner of the tunnel board. Test samples were administered i.p. at sub-convulsive doses and
192
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
the activity was recorded after 30, 45, and 60 min. The number of the tunnel crossed and the total number of tunnels crossed were recorded [15]. 2.5.4. Pentylenetetrazole (PTZ) induced convulsion test Swiss albino mice were divided into five groups (n = 5) and treated with the test samples or saline. PTZ was administered (80 mg/kg s.c.) after 15 min of administration of test samples. Animals were then observed (60 min) for occurrence of seizures and the presence or absence of clonic convulsions. The duration between administration of PTZ and onset of convulsion and mortality, was recorded. An episode of convulsions persisting for a period of at least 10 s was considered as threshold convulsion. Animals devoid of seizures were considered protected [16]. 2.5.5. Effect of diazepam on lethality of crude and processed nux-vomica in mice Diazepam (5 mg/kg, i.p.) was injected into groups of mice. 30 min later the LD100 dose of test samples was injected i.p. The mice were observed for the presence of convulsions, nature of convulsions and mortality if any, for 24 h. 2.5.6. Morphine-induced catalepsy in rats Twenty-five Wistar rats were divided in five groups. Group 1 served as control and the rest of the groups were treated with the test samples in order of samples I, II, III, and IV. After half an hour of the treatment with the test sample the animals were administered with morphine hydrochloride (10 mg/kg i.p.). Catalepsy was measured as immobility percentage. Four distinct stages were discernible; Stage I: the animal sat quietly, with no attempt to move, unless pushed gently, Stage II: the animal didn't move even on being pushed, Stage III: the forepaws were gently put on lower retort ring (7.5 cm high) of the catalepsy stand. The animal maintained the posture for 30 s or more, Stage IV: by raising one of the hind limbs and then removing the support, the raised limb continues to remain suspended in the air. Preservation of this posture for 30 s or more was considered the prerequisite for the onset of stage IV of the catalepsy. Once the animal reached the stage IV or the deepest possible stage, it was subjected to the ring test. The cataleptic animal was gently lifted by its tail and placed on the upper ring (40 cm high) of the cataleptic stand in such a manner that only the limbs of the animal rested on the ring. Once placed, the animal was closely observed for a period of 5 min. The total period for which the animal remained perfectly immobile was taken to represent the depth of catalepsy as “immobility percentage”. 2.6. Phytochemical study 2.6.1. Extraction and quantification The total alkaloidal content was determined by a modified B.P. 1980 method. About 0.4 g accurately weighed fine powder of the drug was saturated with dil. ammonia solution and extracted with 60 ml chloroform in a soxhlet apparatus of suitable size for 2 h. The chloroform extract was then treated with four quantities of sulphuric acid (0.1 N; 20 ml). The combined acid solution was washed with chlo-
roform (20 ml). The aqueous solution after filtration was warmed gently over the water bath in a shallow dish, to remove the traces of chloroform. The solution was cooled and diluted to 100 ml with sulphuric acid (0.1 N). 20 ml of this solution was again diluted to 100 ml with 0.1 N sulphuric acid. The extinction of 1 cm layer was measured at 262 nm and 330 nm [17]. 2.6.2. Assay of strychnine in powdered drug The processed seeds were coarsely powdered. They were then boiled in hydrochloric acid (50%; 20 ml) and filtered. After filtration, the aqueous portion was extracted with four quantities of chloroform (25 ml). The aqueous portion was made alkaline with the dilute ammonia solution drop wise and again extracted with four quantities of chloroform (25 ml). The chloroform extract was then combined and evaporated to dryness. The residue so obtained was dissolved in a mixture of sulphuric acid (3%; 15 ml) and nitric acid (2 ml). Few crystals of sodium nitrite were added to the solution. After about 40 min at room temperature, the solution was again made alkaline with 10% sodium hydroxide solution and the alkaline solution was then extracted with chloroform (20 ml). The chloroform layer was washed with sodium hydroxide (5 ml) and then with two quantities of water (10 ml). Each chloroform extract thus processed similarly. The second aqueous washing was titrated with sulphuric acid (0.1 N) using methyl red as indicator. If more than 0.1 ml was required, the chloroform solution was washed further with water until the aqueous washing required not more than 0.1 ml of 0.1 N sulphuric acid for neutralization. The chloroform solution was then dried with the help of anhydrous sodium sulphate and filtered. It was then evaporated to dryness. The residue was dissolved in sulphuric acid (0.1 N; 10 ml) and an excess of acid back titrated with sodium hydroxide (0.1 N). Each ml of sulphuric acid (0.1 N) is equal to 0.03344 g of strychnine. The result was multiplied with 1.02 to correct for the loss of strychinine [18]. 2.6.3. Chromatographic analysis All the samples were chromatographed as crude alkaloid fraction (CAF) on silica gel TLC plate with solvent system (chloroform:methanol; 95:5 v/v) and spray of Dragendorff's reagent was used for detection. All the test drug samples were analyzed for the total alkaloidal content in the different portions of the seeds and total contents of strychnine. 2.7. Statistical analysis The values are expressed as mean ± SEM. Statistical significance was analyzed by ANOVA followed by Dunnet's T test. P < 0.05 was considered to be significant. 3. Results 3.1. Pharmacological studies 3.1.1. LD50 dose determination Nux-vomica extract, when administered i.p. to the animals, showed a great variation in the toxic dose. The results have been summarized in the Table 1.
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195 Table 1 LD50 values (method of Karber) of intraperitonially administered extract and percentage of strychnine in differently processed samples of nux-vomica seed.
Table 4 Effect of crude and processed nux-vomica on PTZ induced convulsions in mice. Groups
S. no.
1 2 3 4
Sample
Crude drug (sample A) Aloe and ginger treated (sample B) Ghrit treated (sample C) Milk treated (sample D)
LD50 value (mg/kg)
Percentage of strychnine (% w/w) Test drug
Seed coat
52
2.1875
0.4336
70
1.29
0.745
205
1.212
0.0525
600
0.4375
1.12
3.1.2. Effect of crude and processed nux-vomica on pentobarbitone-induced hypnosis in mice The mean sleeping time of animals treated with pentobarbital sodium (50 mg/kg i.p.) was 29.8 ± 2.87 mins. Pretreatment of the animals at sub-convulsive doses with the crude and treated samples of nux-vomica enhanced pentobarbitone-induced sleeping time. Milk treated sample showed the maximal potentiation followed by samples C, A and B. Intermittent convulsions were noted in the crude drug sample group (sample A) even while the mice were asleep. Such convulsions were also present in some mice, treated with sample B but were absent in those treated with sample C and D (Table 2). 3.1.3. Effect of crude and processed nux-vomica on spontaneous motor activity in mice The effect of the test drugs on Shillito's tunnel board shows that the crude nux-vomica (sample A) markedly Table 2 Effect of crude and processed nux-vomica at sub-convulsive doses on pentobarbitone-induced hypnosis in mice. S. no
Dose (mg/kg)
Test sample
Sleeping time (min)
1 2 3 4 5
10 ml/kg 20 25 70 125
Control Sample A Sample B Sample C Sample D
29.8 ± 2.87 47.6 ± 3.18⁎⁎ 35.6 ± 6.60⁎ 48.0 ± 3.91⁎⁎⁎ 53.8 ± 2.76⁎⁎⁎
Values are expressed as mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎⁎P < 0.01, ⁎P > 0.05, ⁎⁎⁎P < 0.0001. ANOVA followed by Dunnett's T test signifies the test.
193
Control Sample A Sample B Sample C Sample D
Dose (mg/kg)
Time interval between administration of drug and onset of convulsion (min)
20 25 70 125
6.0 ± 0.92 4.75 ± 1.03⁎ 8.0 ± 0.82⁎ 10.0 ± 0.41⁎⁎ 11.25 ± 0.48⁎⁎⁎
Values are expressed as Mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎P > 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.0001. ANOVA followed by Dunnett's T test signifies the test.
reduced the number of tunnels entered at 30, 45 and 60 min of observation time. A similar reduction was noted with samples C and D at similar observation time. There wasn't a significant difference between the results obtained with mice of C and D group throughout the experiment, though the numbers of tunnel entries were slightly more than that of the crude sample group at 45 and 60 min. There was a gradual decrease in the number of entries with sample A treated group. All the treatments were at sub-convulsive doses (Table 3). 3.1.4. Effect of crude and processed nux-vomica on PTZ induced convulsions in mice The effects of the processed and unprocessed samples were compared with pentylenetetrazole (80 mg/kg i.p.) produced convulsion in the experimental animals. The mean time for the onset of convulsions was 6 min. PTZ induced convulsions were markedly inhibited by sample D significantly and to an insignificant extent by sample C. Crude nux-vomica reduced the time of onset of convulsions. The maximum time for onset was noted with sample D followed by C and B (Table 4). 3.1.5. Effect of diazepam on lethality of crude and processed nux-vomica in mice The effect of administration of diazepam 30 min before the test samples was observed to cause any protection against the 100% lethal dose (LD100) of nux-vomica samples. The LD50 values were calculated previously with the method of Karber. Diazepam completely inhibited the lethal effect of samples A, C and D at their respective LD100 values. There was an incomplete but significant inhibition of mortality induced by sample B. Samples A and B produced intermittent convulsions predominantly tonic in nature, while convulsions of clonic
Table 3 Effect of crude and processed nux-vomica on spontaneous motor activity in mice. Time interval between administration of drug and observation (min)
N
Mean number of tunnels entered A
B
C
D
0 30 45 60
5 5 5 5
25.2 ± 0.8 11.0 ± 0.8944⁎ 8.0 ± 0.4472⁎ 4.8 ± 0.5831⁎
25.2 ± 0.9165 29.4 ± 1.030 13.4 ± 0.6782⁎ 13.2 ± 0.5831⁎
20.8 ± 0.8602 4.0 ± 0.6325⁎ 10.8 ± 0.6633⁎ 10.4 ± 0.7483⁎
21.4 ± 0.9274 6.0 ± 0.4472⁎ 13.2 ± 0.8602⁎ 15.0 ± 0.7071⁎
Values are expressed as mean ± SEM (n = 5). Significant difference from corresponding control value; ⁎P < 0.01, ANOVA followed by Dunnett's T test signifies the test.
194
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
and tonic nature were produced by sample C. Sample D produced convulsions predominantly clonic in nature, followed by few episodes of tonic convulsions. 3.1.6. Morphine-induced catalepsy in rats Morphine (10 mg/kg i.p.) produced marked cataleptic effect in rats. Most of the animals passed phase III of the catalepsy with a mean cataleptic score (MCS) of 77.2. Pretreatment with crude nux-vomica produced a slight but significant potentiation of morphine-induced catalepsy. The treatment with samples C and D antagonized the morphineinduced catalepsy (Table 5). 3.2. Phytochemical studies The different components of the seeds after processing were extracted and the amount of total alkaloids was determined. The results have been compiled in Table 6. The extract obtained from the cotyledons of the differently processed nux-vomica was also chromatographed using chloroform:methanol (95:5 v/v) on a silica gel plate and Dragendorff's reagent as spotting agent. The chromatogram showed the presence of a new spot in the nux-vomica processed with milk. Further isolations and characterizations of the new spot shall be discussed elsewhere. The total strychnine contents were determined using modified B.P. 1980 method (Tables 1 and 6). 4. Discussion In the present study, an attempt was made to analyze the pharmacological efficacy of S. nux-vomica, before and after processing (shodhan) it with the methods quoted in
Table 5 Effect of crude and processed nux-vomica on morphine-induced catalepsy in mice. S. no.
Treatment groups
Immobility (%) Mean ± S.E.
Percentage difference with regard to control group
1 2 3 4 5
Control Sample A Sample B Sample C Sample D
77.20 ± 3.79 85.40 ± 1.77⁎ 0±0 0±0 24.20 ± 10.11⁎⁎
– 10 (+) 100 (−) 100 (−) 68.65 (−)
(+) indicates potentiation; (−) indicates inhibition in the effect.
Table 6 Percentage of total alkaloids in different portions of seeds of processed drug samples. S. no.
Drug
1
Aloe and ginger treated (sample B) Ghrit treated (sample C) Milk treated (sample D)
2 3
Percentage of total alkaloids (% w/w) Cotyledon
Seed coat
Embryo
80.6
15.3
0.2
75
13.3
–
71.6
12.5
0.2
the classical Ayurvedic literature, so that their mutual qualitative relativity can be better understood. Shodhan is one of the important processes described in the ancient literatures of Ayurveda with reference to the use of toxic substances including metals, minerals and plants. It is presumed that the technique was advised and used by the ancient scholars to modify/enhance the pharmacological property and reduce or diminish the poisonous effects of any toxic drug and render it suitable to be used therapeutically. Nux-vomica is rarely used in practice as such, due to high strychnine content being opisthotonous in the modern system of medicine, although it is widely used in the alternative system of medicines even today, since they have their own ways of formulating and handling the toxic drugs. The drug is found to be useful in the urinary incontinence of children and elderly when it is due to a relaxed or paralyzed sphincter. It stimulates the sexual organs and hence given with varying success in impotence, spermatorrhea, and sexual frigidity of women. Nux-vomica is one of the best agents for so-called chronic G.I. ailments of various types and origins, but all of an atonic character. It is an important remedy for atony and relaxation of the stomach and bowels and disorders dependent thereon. The comparative neuropharmacological studies were planned keeping in view the clinical use of treated nuxvomica seeds in Ayurveda. Processing of the seeds effectively affected the toxic dose as exhibited in the Table 1. The difference in toxic doses may be attributed to change in total alkaloidal contents and strychnine. Nux-vomica showed significant pentobarbitoneinduced hypnosis potentiating action. However, animals of the crude nux-vomica group (sample A) showed convulsions during sleep that were found to be absent in the animals treated with processed nux-vomica. In addition, the observations indicate that the processings apart from reducing the toxicity, potentiated the barbiturate induced hypnosis. It was also seen that the seeds treated with milk (sample D) contains less amount of strychnine as compared to others, but has a much better response than other samples. This is indicative of the fact that nux-vomica after processing tends to exhibit a CNS depressant action, at sub-convulsive doses. All the samples were found to reduce the locomotor activity of the animals in the Shillito's tunnel board test. Since, exploratory behavior is an inherent attribute of this experimental model, the results indicate that in sub-convulsive doses, crude as well as processed (shodhit) nux-vomica inhibited the exploratory behavior which is again indicative of CNS depressive type action. PTZ is a convulsant known to act on the reticular activating system of brain stem and also on motor cortex. PTZ convulsions are used to evaluate anti-epileptic drugs likely to be used in petitmal epilepsy. Crude nux-vomica (sample A) hastened the onset of PTZ convulsions as was expected, both of them (PTZ and nux-vomica) being established convulsive agents. On the contrary, all the processed samples increased the time of onset of convulsions, and the maximum delay in onset was seen with sample D where the convulsions were inhibited to a significant duration. The results indicate that processing the seeds with milk causes the reversal of the convulsive effect of nux-vomica. Processing with milk imparts the maximal anticonvulsant effect. This
C.K. Katiyar et al. / Fitoterapia 81 (2010) 190–195
action may be attributed to the lesser strychnine content and other chemical transformations that would have occurred during the processing. Catalepsy is known to be associated with rigidity of skeletal muscles brought about by increased motor tone, subsequent to striatal dysfunction. The fact that processed samples exert an anticataleptic action (Table 5) can justify the use of nux-vomica in clinical condition of Parkinson's disease in which muscular rigidity is a sign. Diazepam is clinically used as an antidote in strychnine poisoning. The effect of diazepam (5 mg/kg i.p.) was observed on the mortality induced by crude and processed samples when administered 30 min before the administration of test drug samples. The doses of all the test samples chosen were the 100% lethal doses (LD100). Diazepam completely inhibited the lethal effects of samples A, C and D. There was an incomplete but significant inhibition of mortality induced by sample B (200 mg/kg i.p.). Crude nux-vomica (sample A) and sample B produced intermittent convulsions which were predominantly tonic in nature. Animals treated with sample C also exhibited intermittent convulsions which were clonic and tonic in nature, whereas with sample D the convulsions were predominantly clonic, followed by episodes of tonic convulsions. Experimental catalepsy is regarded as a laboratory counterpart of clinical Parkinsonism. Morphine catalepsy has been investigated and found to be mediated through striatal cholinergic mechanisms [19,20]. The unprocessed nux-vomica extract (sample A) produced a potentiating effect whereas the processed samples inhibited morphine-induced catalepsy (Table 5). The quantitative studies on alkaloids suggest that the content of alkaloids decreases in order from sample A > B > C > D. The order is similar for strychnine content also (Tables 1 and 6). In the morphine-induced catalepsy, the results show that treatment with sample A produces slight potentiation of the morphine-induced catalepsy, while samples B and C completely antagonized this effect and the sample D which earlier exhibited the maximum potentiation in the hypnosis potentiation could not reduce the morphine-induced catalepsy to a significant extent. These studies suggest that the individual cases of treatment cannot be correlated merely on the basis of strychnine contents and neither can it be assumed to be due to spinal motor neuron stimulatory effect of the alkaloid. From these experiments, it was inferred that nux-vomica after processings tends to suppress spontaneous movements, reduces initiative and interest in environment and there was some potentiation of hypnosis. Therefore, it may be concluded that the shodhan treatment brings about some phyto-
195
chemical changes that in turn reverses the pharmacological profile of the drug. The present investigation for the first time, reports that that there is a weak to moderate degree of CNS depressant activity that justifies the use of the processed drug clinically in various neurological disorders like Parkinson's disease and Trigeminal neuralgia for which satisfactory treatment is still awaited. This study also provides an indication of which method to use to attain the desired efficacy and results among those quoted in the texts. However, these pharmacological changes justify the need of detailed phytochemical investigations with reference to changes in phytochemical composition responsible for transformed biological activity. Acknowledgements CK thankfully acknowledges the Dean, Faculty of Ayurveda Head, Department of Rasashashtra and Head, Department of Pharmacology, IMS, BHU for providing the necessary facilities, help and guidance during the tenure. References [1] Bhavamisra. Bhavprakash. Varanasi. Chaukhambha Sanskrit Sansthan; 2002. p. 568. [2] Shashtri MU. Prayogatmak Abhinav Dravyaguna Vigyanam. Patna: Baidyanath Ayurved Bhawan; 1991. p. 266. [3] Gaud SD. Shankar Nighantu. Jabalpur: Banaushadhi Bhandar 1935:96. [4] Singh RS. Vanaushadhi Nirdishika. Lucknow: Uttar Pradesh Hindi Sansthan 1983:1089. [5] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas 1979:679. [6] Anonymous. The Ayurvedic formulary of India (AFI), part I. Ministry of Health and Family Planning, Dept. of Health, Govt. of India, Delhi; 1978. p. 362. [7] Sharma R. Arogya Prakash. Patna: Baidyanath Ayurved Bhawan, 21st ed.; 1982. p. 87. [8] Vaidya B. Nighantu Adarsh. Varanasi: Chaukhambha Sanskrit Sansthan, 2nd ed.; 1978. p. 62. [9] Krishnanandji. Rasatantra Sāra Evam Siddha Prayoga Sangraha. Kaleda, Ajmer: Krishnagopal Ayurved Bhavan; Part I, 8th Edition; 1999. p. 75. [10] Shashtri L. Yogaratnakar. Varanasi: Chaukhambha Sanskrit Sansthan, 7th ed.; 1999. p. 168. [11] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas; 1979. p. 174–5. [12] Sharma S. Rasatarangini. Varanasi: Motilal Banarasidas; 1979. p. 176–7. [13] Ghosh MN. Fundamentals of experimental pharmacology. Kolkatta: Bose Printing House; 2005. p. 197. [14] Ramirez TED, Ruriz NN, Arellano JDQ, Madrigal BR, Michel MTV, Garzon P. J Ethnopharmacol 1998;61:143–52. [15] Shillito EE. Br J Pharmacol 1970;40:113. [16] Adzu B, Amos S, Muazzam I, Inyang US, Gamaniel KS. J Ethnopharmacol 2002;83:139–43. [17] Anonymous. British Pharmacopoeia- II. Cambridge: University Press; 1980. p. 564. [18] Anonymous. British Pharmacopoeia- II. London: General Medical Council Pharmaceutical Press; 1958. p. 510. [19] Bose R, Bhattacharya SK. Indian J Med Res 1979;70:281–8. [20] Bose R, Kumar A, Bhattacharya SK. Neurosci Lett 1979;14:115–8.