Comparative physico-chemical profile of Gunja (Abrus precatorius Linn.) seeds processed through water and Nimbu Swarasa (lemon juice).

Gunja (Abrus precatorius Linn.), known as Indian liquorice, is reputed as one of the world's most deadly but most beautiful seed belonging to the family Fabaceae, characterised under the Upavisha (semi-poisonous drugs) and used extensively in various Ayurvedic formulations with great therapeutic significance. Ayurveda recommended the administration of Gunja only after proper Shodhana (purification procedures) in different media such as Godugdha (cow's milk), Kanji (sour gruel), etc., Apart from the classical methods, some traditional practitioners use Nimbu Swarasa for the Shodhana of Gunja seeds. In this study, an attempt has been made to carry out Shodhana of Gunja seeds using Nimbu Swarasa and water. This study revealed differences in physico-chemical parameters of purified samples, in comparison to raw drugs.

Introduction

Gunja , one of the poisonous plants reported in ancient scriptures of Ayurveda, comes under Upavisha category.[1] Gunja is used in treating various diseases such as Indralupta (alopecia), Shotha (edema), Krimi (helminthes), Kustha (skin diseases), Kandu (itching), Prameha (urinary disorders), etc., after being passed through specific Shodhana.[234] The seeds are often used criminally for killing cattle and it is reported that boiling renders the seed harmless.[5]

It is cited in the classics that Visha (poison) becomes Amrita (nectar) after logical administration[6] and the ancient physicians of Ayurveda successfully used this drug in a number of diseases after proper purification in some specific media. Gunja seeds contain various number of alkaloids, steroids, flavones, triterpenoides, proteins, amino acids, etc., among which albumotoxin and abrin are considered as the main responsible constituents for its poisonous effect. with an estimated human fatal dose of 0.1-1 μg/k.[78] Gunja has been reported for its antitumor,[9] anticancer,[3] antispermatogenic,[10] antifertility,[3] CNS (Central nurvous system) depressant and analgesic activity in rat,[3] in treatment of ulcer and skin affections,[8] antidiarrheal and antihelminthic[8] activities.

While going through the literatures, it can be understood that specific medium is used for Shodhana of particular substances and has a three-way effect on the drug, i.e. purification, detoxification, and potentiation.[11] Studies have also shown that the toxic substances present in the plant drug are transferred into the media during the Shodhana process rendering the drug nontoxic.[12] Specific Shodhana procedures have been prescribed for purification of Gunja seeds.[13] Studies showed that Gunja when purified with cow's milk or Kanji, resulted in depletion of toxic alkaloid hypaphorine and abrin.[14] But, studies on effect of Shodhana with Nimbu Swarasa on Gunja seed.

Considering this, the study has been planned to evaluate the impact of Shodhana through Nimbu Swarasa and water on Gunja seeds.

Materials and Methods

Collection of drug

The plant Gunja was identified by expert plant taxonomist with the help of different flora and its mature seed (red variety) was personally collected from surrounding places of Jamnagar, Gujarat in their natural habitat, during the month of November 2011-January 2012.

Selection of seed

Fully matured dry seeds were first kept in a beaker containing water. The seeds those floated on the surface of water or found broken and fade in colour, were rejected. The seeds those settled at the bottom of the beaker, were selected for purification by following procedure mentioned in Vanausadhi Viseshanka.[13]

Ingredients

Ashuddha Gunja seeds 300 g (100 g for each batch)

Nimbu (Citrus medica) Swarasa (juice) 18 L (6 L for each batch).

Principle: Swedana (Boiling).

Preparation of media

Matured fruits of Nimbu were collected from the local market and juice was extracted manually.

Equipment for Shodhana

Stainless steel vessel (20 cm × 30 cm); capacity of 7 L (used as Dolayantra), stainless steel rod (length 28 cm), stainless steel vessel (48 cm × 30 cm × 7 cm); capacity of 3 L, cotton threads 30 cm in length, measuring mug (capacity of 1 L), muslin cloth (45 cm × 45 cm), digital weighing machine, pyrometer, digital induction cooker, stainless steel knife (blade: 15 cm × 2 cm), frying pan (diameter: 20 cm), stainless steel spatula (length: 30 cm), and measuring cylinder (10 ml, 25 ml).

Procedure

Hundred grams of RGS were kept in a muslin cloth and made into a Pottali, which was immersed in a steel vessel that is filled with Nimbu Swarasa.[12] Then the assembly was boiled on an induction cooker for 3 h at 100°C throughout the experiment. Totally, 6 L of Nimbu Swarasa was utilized for one batch throughout the process. After boiling for 3 h, the seeds were taken out from pottali and washed with lukewarm water. Followed by removal of seed coat manually and allowed to dry in shade by placing on a paper sheet. Same procedure was carried out for all the three batches. After proper drying, the seeds were collected and stored in air tight container and labeled as Nimbu Swarasa Shodhita Gunja seeds (NSGS).

Same procedure was followed for the Shodhana of Gunja seed with water (obtained from RO plant) and the final product was labeled as water Shodhita Gunja seeds (WSGS).

Preparation of sample

The RGS and Shodhita Gunja (both NSGS and WSGS) seeds were powdered and passed though mesh no. 60.

Physico-chemical parameters

Assessment of the parameters such as foreign matter, moisture content, ash value, acid insoluble ash, pH with pH paper, water soluble extractive value, alcohol soluble extractive value, foaming index, and swelling index were carried out following standard procedures.[1516]

HPTLC study

Equipment for HPTLC

A HPTLC system equipped with a sample applicator Linomat V sample applicator (CAMAG, 4132 Muttenz, Switzerland) was used for application of samples. CAMAG Scanner III and Win cats 4.02 were used for scanning the plates. CAMAG twin through glass chamber was used for developing the plates.[17]

Chemicals

Precoated silica gel 60 F254 TLC (Thin Layer Chromatography) aluminum plates (10 × 10 cm, 0.2 mm thick), AR grade toluene, ethyl acetate, glacial acetic acid, and methanol were obtained from M/S Merck Ltd. Mumbai, India.

Samples for HPTLC

The extract of all three samples (RGS, NSGS, and WSGS) for HPTLC were made in same process as mentioned below.

Methanolic extract: 2 g of sample was macerated with 20 ml of methanol for 24 h and filtered. Filtrate was concentrated to 5 ml and used for spotting.

The samples were titled as Track-1, Track-2, and Track-3.

Track-1: Methanolic extract of RGS

Track-2: Methanolic extract of NSGS

Track-3: Methanolic extract of WSGS

Mobile phase: Toluene: Ethyl acetate: Glacial acetic acid (6.5:3.5:0.2)

Detection: Spray with Vanillin–H2SO4.

Chromatographic conditions

Application mode: Camag Linomat V

Development chamber: Camag twin through chamber

Plates:

Precoated Silica Gel GF254 plates. Chamber saturation: 30 min

Development time: 30 min

Development distance: 7 cm

Scanner: Camag Scanner III.

Detection: Deuterium lamp, Tungsten lamp

Data System: Win cats software

The developed plate was scanned to obtain densitogram in visible range from 600 nm to 800 nm with 100 nm interval.

Results and Discussion

In this study, Shodhana of Gunja seeds were carried out by traditionally approved method. Each Shodhana procedure was repeated for three times to establish the validation of the pharmaceutical processing. Shodhana of Gunja was performed by the process of Swedana (boiling) in Nimbu Swarasa, for 3 h. same process was followed for Swedana in water (to serve as a control). Principles of Swedana methods are the extraction process where the solvent enters the cells resulting in the swelling of tissues making easy escape of the soluble constituent. The rate of extraction depends mainly on the temperature and concentration gradient across the cell membrane. Rising of temperature increases the concentration gradient across the cell membrane, thereby increase mass transfer of active principles from solid material to the solvent.[18]

During Shodhana of Gunja in Nimbu Swarasa and water, change in colour in Gunja seed m powder media was noticed and it might be due to the removal of colour containing materials from the endosperm of the seeds. The reddish cream colour powder of raw seeds turned into creamish yellow in colour in case of NSGS and ash colour in case of WSGS after Shodhana [Table 1]. It was observed that 83.9% and 91.66% of purified Gunja were obtained after purification in Nimbu Swarasa and water respectively [Table 2]. It might be due to the extraction of more soluble mass from the seeds by Nimbu Swarasa than water.

Table 1

Organoleptic characters of raw, Nimbu Shodhita and water Shodhita Gunja seed powder

Table 2

Effect of Shodhana after Shodhana with Nimbu Swarasa (lemon juice) and water

The moisture content of NSGS was comparatively lower than the raw and WSGS. Excess of moisture in a sample may encourage the growth of microbes. Lower value of moisture content indicates less chances of microbial growth.[16] Ash value was decreased in both the samples after purification. Ash values in Nimbu Swarasa purified seeds were comparatively less than that of the water Shodhita and RGS. Ash mainly contains inorganic radicals and it should be totally free from carbon particles. Lower the carbon particle in ash reduces the ash value which indicates more purity of a drug. The water soluble extractive value in NSGS was found lower than raw sample but higher than that of the WSGS. It is being observed that all samples are having acidic pH. The pH value was found comparatively lower in NSGS (5.0) than the other two samples [Table 3]. According to some experts, acidic pH indicates Ushnavirya.[18]

Table 3

Physico-chemical parameters of raw and Shodhita Gunja seed

In HPTLC, at short UV 254 nm, huge number of different spots was found in all three samples, which indicates the presence of different components [Table 4]. Presence of one Rf value (0.01) was found in all three samples, which indicates the presence of one common component in all three samples [Figures 1, 7–10].

Table 4

Rf values in short UV (254 nm) of the methanolic extract of all three samples

Figure 1

Short UV 254 nm. (a) Track-1: HPTLC for Methanolic extract of raw Gunja seed. (b) Track-2: HPTLC for methanolic extract of Nimbu Swarasa Shodhita Gunja seed. (c) Track-3: HPTLC for methanolic extract of water Shodhita Gunja seed

Figure 7

HPTLC for methanolic extract of raw Gunja seed (254 nm)

Figure 10

Multiple tracks (254 nm)

At long UV 366 nm, RGS, NSGS, and WSGS showed 6, 11, and 5 spots, respectively. One similar Rf value, (0.01) was detected in all three samples, indicating the presence of one similar compound in all three samples [Figures 2, 4–6 and 11]. Maximum numbers of spots were found in case of NSGS (11 spots), indicating the presence of more components in NSGS than the other two samples (RGS and WSGS) [Table 5]. From the spectral comparison [Figures 12–14], same Rf values were found in case of all three samples, i.e. 0.3, 0.48, and 0.92. From which it can be narrated that the presence of same component is possible in case of all three samples. After spraying with vanillin-H2SO4, RGS, NSGS and WSGS showed 2 (0.71, 0.94), 5 (0.15, 0.60, 0.69, 0.85, 0.94) and 3 (0.60, 0.69, 0.94) spots, respectively [Figure 3].

Figure 2

Long UV 366 nm. (a) Track-1: HPTLC for Methanolic extract of raw Gunja seed. (b) Track-2: HPTLC for methanolic extract of Nimbu Swarasa Shodhita Gunja seed. (c) Track-3: HPTLC for methanolic extract of water Shodhita Gunja seed

Figure 4

HPTLC for methanolic extract of raw Gunja seed (366 nm)

Figure 6

HPTLC for methanolic extract of water Shodhita Gunja seed (366 nm)

Figure 11

Multiple tracks (366 nm)

Table 5

Rf values in long (UV 366 nm) of the methanolic extract of all three samples

Figure 12

UV spectral comparison Rf 0.3 T-1, 2,3

Figure 14

UV spectral comparison Rf 0.92 T-1,2,3

Figure 3

After spraying. (a) Track-1: HPTLC for Methanolic extract of raw Gunja seed. (b) Track-2: HPTLC for methanolic extract of Nimbu Swarasa Shodhita Gunja seed. (c) Track-3: HPTLC for methanolic extract of water Shodhita Gunja seed

HPTLC for methanolic extract of Nimbu Swarasa Shodhita Gunja seed (366 nm)

HPTLC for methanolic extract of Nimbu Swarasa Shodhita Gunja seed (254 nm)

HPTLC for methanolic extract of water Shodhita Gunja seed (254 nm)

UV spectral comparison Rf 0.48 T-1, 2, 3

Conclusion

After Shodhana, changes in physico-chemical parameters of Gunja seeds are observed and more numbers of spots are detected under both 254 nm and 366 nm in case of NSGS, indicating the presence of more number of components in NSGS than the other two samples (RGS and WSGS). However Qualitative estimation of these components, their utility in therapeutics need to be evaluated in further studies.