Effect of detoxification (tadbeer) in content of toxic metabolites of Strychnos nux-vomica: A Unani approach for its use in human.

BACKGROUND: Azaraqi (Strychnos nux-vomica, Loganiaceae) has been the important Unani medicine since long time as a stimulant, anti-inflammatory, and blood purifier. It has been used very frequently by the Unani practitioner. But the Unani system recommends application of azaraqi in medicine only after its detoxification (tadbeer) may be because of the presence of its deadly poisonous alkaloids (strychnine and brucine). In the present investigation, an attempt has been made to quantify the actual content of their toxic alkaloids before and after the tadbeer.
MATERIALS AND METHODS: A sensitive high-performance thin layer chromatographic (HPTLC) method has been developed for estimation of strychnine and brucine in different samples of azaraqi before and after tadbeer. Precoated HPTLC silica gel plates were used as stationary phase and (toluene: Ethyl acetate: Dietylamine 7:2:1 v/v/v) was used as mobile phase. RESULT: The Rf value of strychnine and brucine was found as 0.53 and 0.41, respectively. Detection and quantification were performed by densitometry at 270 nm. The calibration plot was linear in the range of 50-1000 ng of strychnine and brucine, respectively, with the correlation coefficient (r (2)) 0.993 and 0.991 for strychnine and brucine, respectively, which confirms good linearity. The content of strychnine was 0.175, 0.07, 0.18, 0.051, and 0.075% w/w whereas brucine was 0.16, 0.117, 0.061, 0.045, and 0.057 in crude azaraqi, azaraqi without outer cover, azaraqi outer cover only, azaraqi mudabbar and azaraqi mudabbar by fried in ghee, respectively.
CONCLUSION: The detoxification results in sharp decrease in content of toxic metabolites. The process by boiling in milk was found much effective but tedious as compare to frying method.

In Unani System of Medicine, Drugs are classified according to mizaj (temperament). These are Motadil (normal), Derja Awwal (Ist degree), Derja Doem (IInd degree), Derja Some (IIIrd degree), and Derja Chaharum (IVth degree). Some of the drugs have poisonous effects. To minimize the toxic effects, drugs are detoxified, and Amal Tadbeer (detoxification process) is applied. Amal Tadbeer (detoxification process) is a unique process in this system, and drugs are made usable like metals and least toxic when used medicinally. One of the important drugs is Strychnos nux-vomica family. Loganaceae strychnine and brucine are the alkaloids obtained from S. nuxvomica.[1] It was used originally as a nervine tonic and to treat rheumatic pain.[2] Since, the azraaqi is an important ingredient of several Unani and herbal formulations such as habbe azaraqi. The present experimental work was carried out for quantification of strychnine and brucine in different samples before and after tadbeer to confirm, the content of these alkaloids before and after tadbeer (detoxification).

Materials and Methods

Detoxification process

Crude S. nuxvomica seeds (A1) were soaked in water for 7 days. Water was changed daily; the azaraqi is then taken out and washed. Their fleshy outer covering (testa) is peeled off with the knife (A3) and the kernels of azaraqi (S. nuxvomica Linn. fruit kernel) are separated (A2). Remove the embryo part. It is then washed and dried, tied in a clean cloth bag. The bag is immersed in a vessel containing one liter of milk. The milk is then boiled until it evaporates, care being taken that the bag should not touch the bottom of the vessel. Thereafter, azaraqi is removed from the bag and washed thoroughly with water to obtain azaraqi mudabbar (A4), for (A5) sample S. nuxvomica seeds are fried in ghee.[3]

Preparation of samples

One gram of each of the dried sample of azaraqi (A1, A2, A3, A4, and A5) were dissolved in 10 ml of methanol, sonicated for 2 h and left overnight. Then it is filtered and filtrate is collected, the residue left is again dissolved in 10 ml of methanol, sonicated and left overnight, then it is filtered, both the filtrate are mixed and then evaporated to dryness, the residue obtained is then reconstituted in 5 ml of methanol to give 200 mg/ml concentration.

Preparation of standard solution

Five milligrams of strychnine were dissolved in 5 ml methanol to give 1 mg/ml solution. Five milligrams of brucine were dissolved in 5 ml methanol to give 1 mg/ml solution. Different volumes of stock solution with known concentrations of strychnine and brucine (500 μg/mL) in methanol was spotted in duplicate on thin layer chromatography plate in different volumes to obtain concentrations of 50, 100, 200, 250, 500, and 1000 ng/spot, respectively.

High-performance thin layer chromatographic instrumentation

Precoated aluminum sheet (10 cm × 10 cm) with silica gel 60 F 254 of thickness 0.2 mm was used for HPTLC. Different sample were applied in the form of band with the help of linomat 5 applicator attached to high performance thin layer chromatographic (HPTLC) system which was programmed through winCATS, the software which were installed with the apparatus. The quantitative estimation was carried out using single level calibration curve. The standard and samples were spotted in the form of band of 5 mm and chromatogram was developed in the solvent system toluene: Ethyl acetate: Diethylamine (7:2:1 v/v/v). The developed chromatogram was then scanned using CAMAG scanner III at 270 nm using slit dimension 5 mm × 0.30 mm. The data generated by winCATS software were recorded.

Results and Discussion

Several analytical methods for the estimation of strychnine and brucine have been developed.[45678910] Here, the quantitative analysis was performed through HPTLC method using strychnine and brucine as standard marker compound in the S. nuxvomica.

Calibration curve of strychnine and brucine

The chromatogram was developed, dried, and scanned at 270 nm λmax as shown in Figure 1. The Rf value of strychnine and brucine was found as 0.53 and 0.41, respectively. Detection and quantification were performed by densitometry at 270 nm [Figure 2]. The calibration plot was linear in the range of 50–1000 ng of strychnine and brucine, respectively, with the correlation coefficient (r2) 0.993 and 0.991 for strychnine and brucine, respectively [Figure 3], which confirms good linearity.

Figure 1

Developed high-performance thin layer chromatographic plate and chromatogram of standard strychnine and brucine (100 ng/spot), 270 nm wavelength showing Rf value 0.53 and 0.41, respectively

Figure 2

Superimposed ultraviolet spectra of all tracks indicating specificity of the method

Figure 3

Calibration curve for: Standard strychnine (a); standard brucine (b)

Analysis of strychnine and brucine in samples

The content of strychnine was 0.175, 0.07, 0.18, 0.051, and 0.075% w/w whereas brucine was 0.16, 0.117, 0.061, 0.045, and 0.057 in (A1) crude azaraqi, (A2) azaraqi without outer cover, (A3) azaraqi outer cover only, (A4) azaraqi mudabbar, and (A5) azaraqi mudabbar by fried in ghee, respectively. It was observed that higher concentration of strychnine and brucine was present in crude sample whereas the concentration decreases in detoxified sample. The amount present in A4 that is, sample boiled in milk is lower as compare to A5 fried sample. But the boiling process is much tedious and time consuming. The process of frying is easy but the concentration of strychine and brucine was a bit higher as compare to boiling process.

Conclusion

The present study is very useful in optimizing the detoxifying methods used in Unani system of medicines. The detoxification results in sharp decrease in content of toxic metabolites. The process by boiling in milk was found much effective but tedious as compare to frying method.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.