SUMMARY & CONCLUSION

Transcription

1 SUMMARY & CONCLUSION SUMMARY: There are many poisonous plants described in Ayurvedic classics and have been routinely used in medicine after proper purification. Traditional methods of Shodhana procedures (purification) if adopted properly, can transform the poison into nectar. On the other hand, if not purified properly they can cause many a serious adverse reactions even death. Though, these drugs are in use since long, but no evidencebased report is there to prove that these drugs are not having poisonous effect. Among these, one of the most commonly used poisonous plants described in Ayurvedic classical texts is Kupeelu (Strychnos nux-vomica Linn.). If Kupeelu seed is used in crude and unpurified form, it exerts deadly poisonous and even fatal effects due to its reported toxic chemical constituents like strychnine & brucine. It develops restlessness, suffocation, tremors and convulsion gradually and ultimately leading to death due to respiratory arrest within 2-4hrs., if not treated properly. However, after the purification the Kupeelu becomes a very good medicine and is being used widely in a number of formulations as a vital ingredient. This example of Kupeelu satisfies the Charaka s opinion that a deadly poison can become a very good medicine if it is administered properly i.e. used in proper dosage, in proper manner and in the proper stage of the diseases. Literary research on Kupeelu reveals that a very few research work have been carried out on Shodhana (purification) of Kupeelu till date and even the Ayurvedic Pharmacopoeia of India does not provide details regarding Kupeelu. While searching through various research journals, text books of Ayurveda and different search engines it reveals that a very few works have been reported on this aspect of Kupeelu, which draws our attention to carry out a research work based on different Shodhana aspects of Kupeelu with following aims and objectives. AIMS AND OBJECTIVES: The present study was planned to accomplish the following aims & objectives: 1. To carry out a conceptual study related to visa, drug Kupeelu and role of different media used for Shodhana from the available literatures on Ayurveda, modern and other systems of medicine. Shodhana of Kupeelu Page 172

2 2. To carry out Shodhana of Kupeelu seeds as mentioned in different Ayurvedic texts &literatures. 3. To study the characters in pharmacognosy along with phytochemical analysis of the Kupeelu seeds both before and after purification. 4. To assess the acute toxicity, analgesic &anti-inflammatory activity of the test drugs having highest and lowest percentage of strychnine & brucine respectively. This study was carried out in phase-wise manner by incorporating following 5 phases i.e Conceptual study, Pharmacognostical study, Pharmaceutical study, Analytical study and Pharmacological study. CONCEPTUAL STUDY: The toxic plants like Kupeelu, Vatsanabha, Bhallataka etc. are vividly reported in the versatile texts of Dravyaguna along with their different Shodhana procedures. The toxic results induced by these plants and their management are also mentioned within the numerous classics of Dravyaguna. To properly perceive the subject matter of this study, it appears to be necessary to compile the ideas relating to, the Shodhana procedure mentioned in the classics of Ayurveda and to review the literatures concerning the drug employed in the present study. The conceptual study has been carried out in 3 sections: 1) Concept of Visha and Upavisha: Concept of Visha and Upavisha with regards to their terminological derivation, mythological origin, properties, fatal dose, fatal period and systemic effects, etc. have been compiled from various Ayurvedic classics and presented in a systematic manner. The use of Visha dravya in Ayurvedic System of Medicine is available since Samhita period. Brihat Trayee texts describe the types of poisons and their medicinal use in details. The toxic effects of the poisonous plants and therapeutic doses of these plants are clearly elaborated. Depending upon the severity of toxic effects, Visha drugs have been classified as Visha and Upavisha. Upavishas are the substances which exert toxic effects on the body but do not cause instant morbidity. To reduce their toxicity, these Visha and Upavisha drugs are strictly advised to undergo various Shodhana processes prior to their administration in therapeutics. Shodhana of Kupeelu Page 173

3 2) Role of media in Shodhana: The concept concerning Shodhana procedure and role of media for poisonous herbal drugs have been compiled and reported briefly in this section. Previous studies reveal that the media employed in the Shodhana process has very significant role for eradicating the toxic chemical components partially or by transforming them to non-toxic substances. It is also reported that sometimes media act like solvent to dissolve the material for separating them from the insoluble impurities and sometimes media may provide some new organic or inorganic principles to the drug which may be responsible for enhancing their therapeutic efficacy. In the present study the Shodhana of Kupeelu seed was performed by following the principles of Nimajjana, Swedana & Bharjana with various media and the impact of each media on Shodhana of Kupeelu seeds was assessed accordingly. 3) Drug review: In this section, various Ayurvedic and modern literatures and research papers were reviewed regarding the drug Kupeelu (Strychnos nux-vomica Linn.). Historical background, botanical identity, vernacular names, synonyms, rasa panchaka, pharmacological actions, toxicity etc. of the drug were compiled and presented in a systematic manner. The description of the drug Kupeelu was not cited in the Brihat Trayee texts of Ayurveda. Its uses in Ayurveda were recorded from the period of Brinda Madhava (9 th A.D.). Kupeelu has been included under the Upavisha category and the synonyms like Visatinduka, Kupeelu, Visamusti etc., indicate toxic nature of this drug. The toxic alkaloids strychnine and brucine present in it are mainly responsible for its adverse effect like tetanic convulsion. Therefore, it should be administered internally only after Shodhana (purificatory procedures). A good number of compound formulations containing Shodhita seeds of Kupeelu as one of the ingredient have been mentioned in the various classical text books of Ayurveda for the management of different diseases. But no text book of Ayurveda suggests its use in Ayurvedic therapeutics as a single drug entity. Shodhana of Kupeelu Page 174

4 PHARMACOGNOSTICAL STUDY: The whole Pharmacognosy was carried out into three phases as per the standard protocol: 1) Macroscopic study of the raw seeds 2) Microscopic study of the raw seeds 3) Powder characters of the raw & purified seeds Macroscopic study of the raw seeds: It was observed that Kupeelu seeds were mm diameter and 4-6 mm thick. Most of the seeds were regular in shape, disc-shaped, being a little depressed on one side and arched on the other. However, few were irregularly bent and somewhat oval in outline. The edge was rounded or acute. Outer surface of the seeds were ash grey or greenish grey in color, the testa was covered with silky, closely appressed silky hairs radiating from the centre to the circumference. The hilum was situated in the center of either the raised or the depressed surface and a small prominence on the circumference marked the position of the micropyle, which was joined to the hilum by a radical ridge. This line did not exist in the fresh seed, but makes its appearance during the drying and disappeared when the dry seed was soaked in water. Endosperm of the dry seed was hard and horny, but softened when soaked in water; it can then easily be split into two thinner discs, exhibiting the small embryo, consisting of a radicle and two leafy cotyledons, embedded in a copious, grey, translucent, horny endosperm. A narrow slit like cavity was present in the center just beneath the endosperm. A small embryo with two cordate cotyledons and a cylindrical radical was embedded in the grey colored endosperm. The cotyledons were about 5-6 mm long and the radical about 4 mm. Fruit pulp smelled slightly acidic but the dried seeds and their powder were odorless. The raw Seeds were acrid, very bitter in taste; this is much stronger in the kernel than in the investing membrane. Each Nux vomica seeds weighed from gm. Microscopic study of the raw seeds: The transverse section showed a very thin testa consisting of collapsed parenchyma and an epidermal layer of very characteristic lignified hairs (trichomes). The trichomes had a very large, thick walled base with slit like pits. Surface irregularities in base of the trichomes caused them interlock with one another. The Shodhana of Kupeelu Page 175

5 upper portions of the trichomes were found to be set almost at right angle to the bases and all radiated out towards the margin of the seed, giving a characteristic silky appearance of the testa. The upper part of the wall of the trichome was composed of about 10 longitudinal ridges like thickenings united by a thin wall so that the lignified ribs readily separate from one another on powdering. The endosperm consisted of large, thick walled cells, which were composed mainly of hemicelluloses and swelled considerably in boiled water. Fixed oil was present in the meshes of protoplasm with a few alleurone grains and the alkaloids strychnine and brucine. Strychnine was mainly found in the inner part of the endosperm and brucine in the outer layers. Powder characters of the raw & purified seeds Raw powdered Nux-vomica seed was pale brown to yellowish gray in color; consisting chiefly of thick-walled endosperm cells containing globules of a fixed oil and a few small aleurone grains, fragments of strongly lignified, non-glandular hairs, the walls of the latter possessing large, circular, or long, slit-like pores. In the tissues of the adhering pulp there were a few small, nearly spherical starch grains. In Microscopic study of the powdered samples, most of the characters of raw Kupeelu seeds were observed in Shodhita Kupeelu samples. Only the aleurone grains were found to be absent in the powder microscopy of all the Shodhita samples. Other characters were found to be similar to the powder drug of the raw samples. PHARMACEUTICAL STUDY: In this phase of the study, an attempt has been made to validate the different Shodhana procedures of Kupeelu with various media like Gomutra (cow urine), Godugdha (cow milk), Goghrita (cow ghee), Kanji (sour gruel), Eranda taila (castor oil), Ardraka swarasa (fresh ginger juice) and water as control. To provide a high degree of accuracy to the Shodhana procedures, validation of each process was carried out in three different batches and the results were documented accordingly. Ι. Shodhana with Gomutra (cow urine): Reference of the Shodhana procedure: Rasatarangini, Principle: Nimajjana (Dipping) Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; Gomutra: 7 lit (1lit 7 days) ΙΙ. Shodhana with Godugdha (cow milk): Reference of the Shodhana procedure: Rasatarangini, Principle: Swedana (Boiling) Shodhana of Kupeelu Page 176

6 Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; Godugdha: 6 lit. ΙΙΙ. Shodhana with Goghrita (cow ghee): Reference of the Shodhana procedure: Rasatarangini, Principle: Bharjana (Frying) Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; Goghrita: 25 ml. ΙV. Shodhana of Kupeelu with successive two media first Gomutra (cow urine) followed by Godugdha (cow milk): Reference of the Shodhana procedure: Siddhayoga Samgraha, Principle: Nimmajana (Dipping) & Swedana (Boiling), Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; Gomutra: 7 lit.(1lit. 7 days); Godugdha: 5 lit. V. Shodhana of Kupeelu consecutively with Gomutra (cow urine), Godugdha (cow milk) and Goghrita (cow ghee): Reference of the Shodhana procedure: A.F.I, Principle: Nimmajana(Dipping), Swedana (Boiling) and Bharjana (Frying), Ingredients:AshuddhaKupeelu seeds (KR): 100 g. ;Gomutra: 7 lit.(1lit. 7 days) ;Godugdha: 5 lit. ; Goghrita: 20 ml. VΙ. Shodhana with Eranda taila: Reference of the Shodhana procedure: Dhanwantari Banausadhi Visesanka, Principle: Bharjana (Frying), Ingredients: Ashuddha Kupeelu seeds (KR) : 100 g; Eranda taila(castor oil): 20 ml. VΙΙ. Shodhana with Kanji (sour gruel): Reference of the Shodhana procedure: Rasatarangini, Principle: Nimajjana (Dipping) Ingredients: Ashuddha Kupeelu seeds (KR): 100 g. ;Kanji: 3 lit. (1lit. 3 days) VΙΙΙ. Shodhana with Ardraka swarasa: Reference of the Shodhana procedure: AduAnjirnaGuno-Vishesatao(A book on traditional practice of medicine in Gujarat), Principle: Nimajjana (Dipping), Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; Fresh Ardraka swarasa: 1 lit. ΙX. Shodhana of Kupeelu by dipping in water: Principle: Nimajjana (Dipping), Ingredients: Ashuddha Kupeelu seeds (KR) : 100 g; R.O Water: 7 lit. (1lit. 7 days) X. Shodhana of Kupeelu by boiling in water: Principle: Swedana (Boiling), Ingredients: Ashuddha Kupeelu seeds (KR): 100 g; R.O water: 6 lit. During Shodhana of Kupeelu with various media, change in color of the media was noticed and it may be due to the removal of color containing materials like tannin Shodhana of Kupeelu Page 177

7 from the seeds. Taste of every media became bitter after Shodhana due to the extraction of bitter principles like strychnine, brucine, etc., from the seeds. Changes in organoleptic characters of Kupeelu seeds were also observed and the final weight obtained after each Shodhana procedure was noted accordingly. It was observed that maximum loss in final yield (49.67%) was found after Shodhana with Goghrita whereas minimum loss in final yield ( 13.53%) was obtained after purification with boiling water. ANALYTICAL STUDY: In this phase of the study, the physicochemical analysis of different samples such as raw and Shodhita Kupeelu seeds was carried out followed by quantitative estimation of their toxic alkaloids viz. strychnine & brucine by HPTLC. The analytical work was carried out in following phases; 1) Physico-chemical analysis, 2) Preliminary phytochemical investigation 3) T.L.C. Analysis. 4) HPTLC Analysis. Differences in all the physico-chemical parameters were observed among the raw and Shodhita Kupeelu samples. In comparison to the raw seeds, moisture content was increased in all the Shodhita samples except in KAS. It indicates more removal of intracellular water portion from the raw seeds by the process of diffusion during the Shodhana process with Ardraka Swarasa. Maximum ash content was found in the Gomutra Shodhita sample and it might be due the addition of more organic and inorganic contents like nitrogen, sulphur, phosphorus, hippuric acid, NaCl etc., from cow s urine to the raw seeds. Water soluble extractives of all the purified samples were found to be decreased in comparison to the raw drug (KR). This might be due to the solubility of the contents like strychnine, brucine, loganin etc., in water. The water & alcohol soluble extractive values of Gomutra purified sample were found to be lowest in comparison to all other samples. The result indicates maximum removal of water & alcohol soluble compounds from the raw seeds by Gomutra. The ph values of all the samples were found to be acidic. It was in the range between (KET-4.51; KGM-6.25). Relatively higher ph was obtained in the Gomutra purified sample which might be due to the alkalinity of cow s urine that neutralized the acidic ph of the raw drug to some extent. Qualitative tests revealed the presence of Shodhana of Kupeelu Page 178

8 alkaloids; oil, carbohydrate, protein and tannin are present in raw sample as well as purified samples. Steroid, Flavonoid are absent in raw and the purified samples. Glycoside was found to be absent in all the purified samples in comparison to the raw drug. It might be due to the hydrolysis of loganin into an aglycone loganetin, or converted into secologanin. HPTLC profiles of raw and Shodhita Kupeelu indicate that some peaks disappeared and some new peaks appeared after Shodhana processes. In raw sample, total 4 peaks were found whereas 3-8 peaks were observed in the purified samples under 254 nm. This disappearance and newly appearance of peaks suggest the extraction of some components like strychnine, brucine, loganin etc., and formation of some new compound during Shodhana process. The Rf values of strychnine and brucine standard were found as 0.54 & 0.34 respectively which were also present in all the samples. In quantitative H.P.T.L.C study, decrease in strychnine and brucine content was found in all the Shodhita samples when compared to the raw drug. Strychnine and Brucine contents were found to be lowest in the sample purified by Gomutra-Godugdha (KGMD) when compared to the other samples. It might be due to the reason that during Shodhana processes, some amount of Strychnine and Brucine were removed by diffusion process into cow s urine. Further boiling in cow s milk also initiated more diffusion of the alkaloids from the seeds as well as some amount of Strychnine and Brucine might have been converted into their N-oxidative derivatives with lesser toxicity. PHARMACOLOGICAL STUDY: The pharmacological study was carried out in three phases i.e., acute toxicity study, anti-inflammatory activity study and analgesic activity study. Wistar strain albino rats of either sex; weighing 210 ± 60 g and Swiss albino mice of either sex; weighing 24 ± 4g were used for the study. The experiments were carried out in conformity with the Institutional Animal Ethics Committee (IAEC) after obtaining its permission (IAEC 06/09-11/PhD/04). In the Pharmacological study, Kupeelu seeds subjected to two classical methods of Shodhana (i.e., Kanji and A.F.I methods), were evaluated for analgesic and antiinflammatory activities to know whether classical purificatory procedure affect the efficacy or not i.e., whether efficacy will remain same or it will enhance the efficacy or it will decrease the efficacy. Shodhana of Kupeelu Page 179

9 Acute toxicity study was conducted following the standard procedure as per OECD guideline 425 using limit dose test of Up and down method. Raw Kupeelu (KR) treated animals showed severe convulsions followed by death at the dose of 175mg/kg. Even at the dose of 100mg/kg also, animals showed convulsions. Approximate LD50 of raw Kupeelu (KR) was found to be 175mg/kg and it was between 265mg/kg for Kanji Shodhita sample (KKJ). In contrast to these, LD50 of A.F.I Shodhita sample (KGMDG) was found to be 1098mg/kg. The result suggests that the A. F. I method is more effective than Kanji method in reducing the toxicity of the drug. Anti-inflammatory study was carried out by adopting two methods, Carrageenan induced paw oedema and Formaldehyde induced paw oedema in rats. The result of anti-inflammatory study revealed that the samples KR (i.e., raw seed) & KKJ (i.e., Shodhita with Kanji) having significant anti-inflammatory activity against formalin induced inflammation. However, the test drug KGMDG (i.e., Shodhita with Gomutra- Godugha-Goghrita) non-significantly inhibited both the phases of formalin induced inflammatory response indicates mild action of the test drug on proliferative phases of inflammation. In analgesic activity study the analgesic testing protocol was selected such that both centrally and peripherally mediated effects could be ascertained by adopting formalin induced pain and tail flick response methods. All the three samples were failed to prove any statistically significant analgesic activity against tail flick test. In Formalin induced pain response test, the drug KGMDG was only found to be statistically significant when compared to the control, which indicates the presence of marked analgesic activity in the drug mediated through modulation of neuropeptides. Shodhana of Kupeelu Page 180

10 CONCLUSIONS: Basing upon the results observed during present study following conclusions may be drawn: Review of literature reveals that In Ayurveda, Brihat Trayee texts do not provide any information regardingkupeelu.its usage in Ayurvedic formulations started from the period of Brinda Madhava (9 th A.D.). A good number of compound formulations containing Shodhita Kupeelu seeds have been reported in Ayurveda mainly for the treatment of Agnimandya, Amavata, Shula, Krimi, Vatavyadhi etc. Kupeelu has not been recommended by any author for its use in Ayurvedic therapeutics as a single drug entity. In Powder microscopy, aleurone grains were found to be absent in all the Shodhita samples when compared to the raw & purified samples. This observation can be used as an index for differentiating the purified seeds from the raw one. Sodhana procedures altered the organoleptic characters of the samples in comparison to the raw Kupeelu seeds. Based on the yield of final product, Eranda taila (castor oil) may be considered as better media for Shodhana in respect to others. Regarding the utilization of media during the Shodhana, only Ardraka Swarasa shows complete amalgamation during the process. Recovery of media was found to be nil after completion of this process. Goghrita & Eranda taila may be considered as the better media for Shodhana when compared to the others on the basis of easy applicability, time saving and cost effectiveness. Gomutra may be considered as better extraction media to raw Kupeelu seeds as the smallest amount of water & alcohol soluble material has been found in the Gomutra Shodhita sample. Glycoside loganin was found to be absent in all the Shodhita samples indicates qualitative changes in the drug during the Shodhana process. Highest reduction in the toxic strychnine & brucine contents was observed in the Gomutra-Godugdha procedure (first dipping in Gomutra followed by boiling in Godugdha) of Kupeelu Shodhana. Hence, it may be concluded that this Shodhana Shodhana of Kupeelu Page 181

11 method is the best method among other processes where reduction of the toxic alkaloids are concerned. There was an increase in LD50 of Shodhita samples in comparison to the raw drug. Again the LD50 of Gomutra-Godugha-Goghrita purified sample (KGMDG) was higher than that of Kanji purified sample (KKJ). Hence, it can be concluded that A. F. I recommended method is more effective than Kanji method in reducing the toxicity of the drug. In the Pharmacological study, Kupeelu seeds purified with Gomutra-Godugha- Goghrita (A.F.I method) showed significant analgesic activity through suppressing neurogenic pain, while the seeds purified with Kanji revealed better antiinflammatory activity. Hence, it can be suggested that for the management of acute inflammatory conditions Kanji shodhita samples can be preferred, while to treat acute pain, Gomutra-Godugha-Goghrita (A. F. I method) Shodhita sample can be utilized. However, further detailed pharmacological studies in different experimental models are required. Shodhana of Kupeelu Page 182