Introduction:

Kupeelu (Strychnos nuxvomica Linn), a well known plant in Indian system of medicine is being used extensively in different classical formulations with great therapeutic significance. Though the plant is described under the ‘Upavisa Vargas’ (sub poisonous group) (1), it’s seeds have been used successfully in different formulations to combat different diseases after proper Samaskar known as Shodhana (processing or purification) (2). The seeds are mainly used as aphrodisiac, appetizer, anti-periodic, digestive, purgative, and stimulant. Further the seeds are also used in anemia, asthma, bronchitis, intermittent & malarial fever and in weakness of extremities (3). Nux-vomica was introduced in Europe in the sixteenth century, but was not much used in medicine, being chiefly employed to poison dogs, cats, crows, etc (4). It is claimed in the ancient manuscripts of Ayurveda that the ‘Visha’ becomes ‘Amrita’ after logical use (5) and the physicians of Ayurveda successfully employed this drug in a number of diseases after proper purification in some specific media.

The concept of Shodhana (processing or purification) in Ayurveda is not only a process of purification/detoxification but also a process to enhance the potency and efficacy of the drug (6). It is reported that Aconite (Vatsanabha) purified by cow’s urine is converted to cardiac stimulant, whereas raw Aconite is cardiac depressant (7). Purified Kupeelu is also claimed to be a potent drug in countering old age problems and specially recommended during senility as Rasayana (antioxidant) (8).The plant is also found to have analgesic & anti-inflammatory (9), anti-oxidant (10), anti-tumor (11), anti-snake venom (12), anti-diarrhoeal (13) and hepatoprotective (14) activities when studied in animal models.

In a previous study, 16 alkaloids have been seperated and identified from the crude nux vomica and 80% of them are Strychnine and Brucine, as well as their derivatives such as isoStrychnine and Brucine N-oxide (15). Strychnine (C₂₁H₂₂O₂N₂; m.p. 286 to 288⁰C) and Brucine (C₂₃H₂₆O₄N₂; m.p. 178⁰C) have been reported as the most important and strongly toxic alkaloids present in this, besides other minor alkaloidal constituents (16). It is also reported that Nux-vomica in large doses, producing tetanic convulsions and eventually death and in lesser doses it may manifest mental derangement (17). So it is mandatory to purify or properly processed Nux-vomica seeds prior to its administration in therapeutics. There are also few reports of previous research works advocating a variety of methods of purification of Nux-vomica seeds as per Chinese (18), Unani (19) and Ayurveda (20) system of medicine. However, the methods of purification and analytical techniques were different from the present study. The purpose of this study was to evaluate the role of purification on the quantitative reduction of toxic alkaloids of Kupeelu seeds by HPTLC technique. Therefore the present study was planned to find out the effect of Shodhana (purification) with Eranda taila (castor oil) on the quantitative reduction of toxic Strychnine and Brucine contents in nux vomica seeds.

Materials and methods

Collection of drugs

Fully matured Kupeelu (Strychnos nuxvomica Linn.) fruits were collected from the field of Bankura district, West Bengal in India during the month of December and were botanically authenticated by pharmacognosists and sample specimen were kept in the museum for future reference. Seeds were taken out from the fruit pulp, thoroughly washed in tap water and shade dried.

Selection of seeds (14)

The dried seeds were first dropped in a beaker containing water. The seeds which floated on the surface of water or found broken, black in color were rejected and the seeds which were settled at the bottom of the beaker were selected for purification after drying in air and were considered as raw drug (KR).

Collection of media

Eranda taila (castor oil, Brand name: APPU, Manufactured by Shree Western G & C industries, Ahmedabad) was procured from the local market and used for processing the Nux-vomica seeds.

Equipments for Shodhana (Purification)

Frying pan (diameter-20cm), stainless steel spatula (length 30 cm), digital weighing machine and, induction heater.

Equipments for HPTLC

A CAMAG (Switzerland) HPTLC system equipped with a sample applicator Linomat V sample applicator was used for application of samples. CAMAG TLC Scanner 3, Reprostar and Wincats 4.02 were used for scanning the plates. CAMAG twin through glass chamber was used for developing the plates.

Chemicals

Pure Strychnine and Brucine were obtained from Sigma Aldrich, U.S.A and precoated silica gel 60 F₂₅₄ TLC aluminium plates (10×10 cms, 0.2mm thick), AR grade toluene, ethyl acetate, diethyl amine, methanol and chloroform were obtained from M/S Merck Ltd. Mumbai, India.

Method of purification of Kupeelu (Strychnos nuxvomica Linn.)

Purification method was carried out in three batches by frying with Eranda taila (castor oil) as per the traditional process (21) mentioned below:

100g. of seeds were fried with 20 ml. castor oil in mild temperature (temperature was set at 60⁰C) on an induction heater until the seeds became swollen and reddish yellow in color. Seeds were then taken out from the heater, seed coats were removed as much as possible by rubbing them over the fingers and immediately made into powder form. The powdered materials were kept in an airtight glass container and marked as ‘KET powder’ for further use.

HPTLC method for estimation of Strychnine and Brucine

Preparation of standard Strychnine and Brucine solution

Strychnine standard (10 mg) and Brucine standard (10 mg) were accurately weighed and dissolved in methanol in two standard flasks and final volumes were adjusted to 10 ml with methanol. (1 µg/µl)

Calibration curve for Strychnine and Brucine

The standard solutions corresponding to 2µg to 6µg of standard Strychnine and Brucine were applied on TLC plates (10cm× 10cm), precoated with silica gel as 6 mm bands by using CAMAG Linomat ΙV sample applicator. The plate was developed in a solvent system of Toluene: Ethyl acetate: Diethyl amine (7: 2:1, v/v) in a CAMAG twin through chamber up to a distance of 7.5 cm at a temperature of 30 ± 2⁰C. The plates were air dried and scanned at a wavelength of 254 nm using CAMAG TLC scanner and CATS V 4.06 software. The peak area of Strychnine and Brucine were recorded for each concentration. The calibration curves of Strychnine and Brucine were obtained by plotting the graphs of peak areas vs. concentrations of Strychnine and Brucine.

Preparation of sample solutions for estimation of Strychnine and Brucine

The raw & the purified samples (2g. each) both were defatted individually with petroleum ether. Defatted samples were then mixed with 10% ammonia and finally extracted with 25 ml methanol for 1 hr. under reflux. The methanol extracts were filtered and concentrated to 5 ml and used as test solutions. 5µl of each test solution was spotted along with 2 to 6 µl standard solutions of Strychnine and Brucine. The plates were developed in mobile phase of Toluene: Ethyl acetate: Diethyl amine (7:2:1, v/v) and scanned at 254 nm for Strychnine and Brucine. Peak areas were noted and quantity of Strychnine and Brucine were calculated by comparing the areas of standard solutions from calibration curve.

Results and Discussion

It was observed in the organoleptic study that the greyish powder of the raw seeds turned into reddish brown in colour after frying in Eranda (castor) oil and pungent smell was coming out from the purified seeds. The organoleptic characters of raw and purified samples were tabulated in table no.1. While carrying out the preliminary phytochemical investigations the presence of alkaloids, tannins, carbohydrates, proteins, fixed oils were detected in methanolic extracts of raw and purified seeds (table no.2). The presence of Strychnine and Brucine was confirmed by comparing the Rf values with that of standard markers by HPTLC. Both the samples were evaluated for physicochemical parameters like loss on drying, total ash, Ph value, water soluble extractive, alcohol soluble extractive etc. and the results were put into table no.3.

In HPTLC chromatogram, UV spectrum at 254 nm of standard Strychnine (R_f 0.54) and standard Brucine (R_f 0.34) were shown in Figure 1-2 and peak areas of Strychnine and Brucine in both the samples were exposed in Figure 3-4. Calibration curves of Strychnine and Brucine were prepared by plotting concentrations of Strychnine and Brucine in the range of 2-6 µg/spot versus average area of the peak. The responses for concentrations of standard Strychnine and Brucine were found to be linear (Figure-5 & Figure-6). The amount of Strychnine and Brucine in raw & purified samples were computed from the calibration curves which suggests the reduction of Strychnine and Brucine content by 67.36% and 46.97% respectively in the castor oil purified sample (table no.4). It might be due to the fact that some amount of Strychnine and Brucine might had been converted into less toxic derivatives like isostrychnine, isobrucine, Strychnine N-oxide, Brucine N-oxide etc. during the frying process in Eranda taila. Although further more study is required to explore the exact reason for decreasing the Strychnine and Brucine content.

Conclusion

From this study it may be concluded that castor oil (Eranda taila) is an effective media for purification of Kupeelu seeds as far as toxic alkaloids are concerned. The findings strongly confirm the claims of the traditional practitioners of Ayurveda that Shodhana (proper processing) of Kupeelu by Eranda taila successfully reduces the toxic elements of the drug. The method was found to be very simple, less time consuming and cost effective also.

Acknowledgement

The authors are very much thankful to Director of I.P.G.T & R.A, G.A.U, Jamnagar, India for providing all necessary facilities in carrying out this work.

References:

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18. Cai BC, Hattori M, Namba T. Processing of nux vomica. II. Changes in alkaloid composition of the seeds of Strychnos nux-vomica on traditional drug-processing. Chem Pharm Bull [Internet].1990[cited2010June3];38(5):1295-1298. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2393954

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Table No.1: Organoleptic characters of raw & purified Kupelu seeds powder

Parameters	Organoleptic *characters of raw Kupelu* seeds powder (KR)**	Organoleptic *characters of Kupelu* seeds powder purified by Eranda taila (KET) in three batches**
Parameters		Batch 1	Batch 2	Batch 3
Texture	Smooth	Smooth	Smooth	Smooth
Colour	Greyish white	Reddish brown	Reddish brown	Reddish brown
Odour	Slightly acidic	Pungent	Pungent	Pungent
Taste	Intense bitter	Bitter	Bitter	Bitter

Table No.2: Qualitative tests for various functional groups

Functional group	Test	Raw drug (KR)	Purified drug (KET)
Carbohydrate	Molish’s test	+ ve	+ ve
Protein	Precipitation test with 5% lead acetate , 5% CuSO₄ and 5%ammonium sulphate solution	+ ve (white colloidal ppt. obtained)	+ ve (white colloidal ppt. obtained)
Oil	Filter paper test	+ ve (Filter paper gets permanently stained with oils)	+ ve (Filter paper gets permanently stained with oils)
Tanin	5% Fecl₃ test	+ ve	+ ve
Steroid	Libermann-Buchard test	- ve	- ve
Alkaloid	Dragendorff’s test	+ ve	+ ve
Alkaloid	Wagner’s test	+ ve	+ ve
Flavonoids	Lead acetate test	- ve	- ve

Table No.3: Physicochemical parameters of raw and purified seeds

Parameters	Samples
Parameters	Raw Kupeelu	Purified by Eranda taila (Castor oil)
Loss on drying	3.39 % w/w	4.03 % w/w
Ash value	1.11% w/w	1.07 % w/w
Water soluble extractive	37.83 % w/w	32.78 % w/w
Methanol soluble extractive	3.89 % w/w	7.72 % w/w
pH	5.75	4.51

Table No.4: Results of estimation of Strychnine and Brucine in raw and purified samples of Kupeelu by HPTLC

Samples

Amount of Strychnine found (% w/w)

Amount of Brucine found (% w/w)

Raw Kupeelu (KR)

1.44

0.66

Kupeelu purified by Eranda taila (KET)

0.47

0.35

Figure 1: HPTLC profile of standard Strychnine

Figure 2: HPTLC profile of standard Brucine

Figure 3: HPTLC of raw Kupeelu showing peak area of Strychnine and Brucine

Figure 4: HPTLC of Kupeelu purified by castor oil showing peak area of Strychnine and Brucine

Figure 5: Calibration curve of Strychnine

Figure 6: Calibration curve of Brucine

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