Physicochemical standardization and drug analysis of anindigenous formulation: Asanadi kashaya

Introduction

The modification done to the drug considering its usage is known as kashaya kalpana.¹Kashaya is one among the panchavidha kashaya kalpana viz. Svarasa, Kalka, Kashaya, Hima and Phanta. It is prescribed in practice depending upon the strength of disease and the diseased individual. In addition to this, it does depend upon the type of drug as well. Conventionally, the kashaya is prepared by adding sixteen parts of water in a part of drug and is reduced to one eighth. ² On the other hand, Kashaya, now a day, is prepared commercially at a large scale and is preserved in a bottle for a longer period of time by adding the standard preservative. Since modification in the method of preparation transforms the qualities of a substance and results in the change of its action,³ dilution and addition of preservatives is Introduction The modification done to the drug considering its usage is known as kashaya kalpana.¹Kashaya is one among the panchavidha kashaya kalpana viz. Svarasa, Kalka, Kashaya, Hima and Phanta. It is prescribed in practice depending upon the strength of disease and the diseased individual. In addition to this, it does depend upon the type of drug as well. Conventionally, the kashaya is prepared by adding sixteen parts of water in a part of drug and is reduced to one eighth. ² On the other hand, Kashaya, now a day, is prepared commercially at a large scale and is preserved in a bottle for a longer period of time by adding the standard preservative. Since modification in the method of preparation transforms the qualities of a substance and results in the change of its action,³ dilution and addition of preservatives is also believed to catalyze the effect of medicine. Currently, both the type of preparations are in practice though traditional practice advocates freshly prepared kashaya to be more effective than the preserved one. Since there is a need to evaluate the variations in different parameters of freshly prepared kashaya and kashaya prepared with preservatives with enhanced shelf life, following study is carried out.

Aims and Objectives: Standardization and HPTLC of freshly prepared kashaya and kashaya prepared with preservatives with enhanced shelf life

Materials and Methods

Material Collection: Asanadi Kwatha churna (a coarse powder), of same batch, is procured from the pharmacy of Sri Dharmasthala Manjunatheshwara college of Ayurveda and Hospital, Hassan, Karnataka, India (a GMP certified company).

Extract Preparation: For freshly prepared kashaya, a part of Asanadi Kwatha churna was taken, boiled in eighteen parts of water and was reduced to one eighth of that of original volume. Whereas, the commercial kashaya was prepared from the same ingredients in the pharmacy of Sri Dharmasthala Manjunatheshwara college of Ayurveda and Hospital, Hassan, Karnataka, India and sodium benzoate is added as a preservative. It was packed in a bottle until it is used.

Estimation of Physico-chemical parameters

A) Refractive Index: A drop of water was placed on the prism, drive knob was adjusted in such a way that the boundary line intersects the separatrix exactly at the centre and the reading was noted. Distilled water has a refractive index of 1.3325 at 25˚C. The difference between the reading and 1.3325 gives the error of the instrument. If the reading is less than 1.3325, the error is minus (-) and the correction is plus (+). However, if the reading is more the error is plus (+) and the correction is minus (-). Refractive index of oil was determined using 1 drop of the sample. Refractive index of the test samples were measured at 28˚C.

B) Specific gravity: A specific gravity bottle was cleaned by shaking with acetone and then with ether. The bottle was dried and the weight was noted. Sample solution was cooled to the room temperature. The specific gravity bottle was carefully filled with the test liquid. The stopper was inserted, surplus liquid was removed and the weight was noted. The procedure was repeated using distilled water in the place of sample solution.

C) Viscosity: The given sample is filled in a U tube viscometer in accordance with the expected viscosity of the liquid so that the fluid level stands within 0.2 mm of the filling mark of the viscometer when the capillary is vertical and the specified temperature is attained by the test liquid. The liquid is sucked or blown to the specified height of the viscometer and the time taken for the sample to pass the two marks is measured. Viscosity is measured using the formula

η1= ρ1t1 X η2 ρ2t2

η1 – Viscosity of sample

η2 - Viscosity of water

t1 and t 2- time taken for the sample and water to pass the meniscus

ρ1 and ρ2 – Density of sample and water

X= Specific gravity of sample x 0.9961/specific gravity of water 𝞟= XxTime for samplex1.004/specific gravity of waterx70sec

D) Total solids: Accurately weighed 50 g of the sample was transferred to an evaporating china dish, was dried to a constant weight, evaporated to dryness on a water bath and dried at 1050C for 3 hours. After cooling the dish containing the residue in a desiccator for 30 minutes, it was weighed immediately.

HPTLC: 10ml of freshly prepared Asanadi kashaya and preservative added Asanadi kashaya samples were partitioned with 20 ml butanol in a separating funnel and kept for 24 hours. The butanol fraction was collected and filtered. The butanol was then made to evaporate on a water bath and it is dissolved in 10.0ml of methanol. 3 and 6µl of the above samples were applied on a precoated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. The plate was developed in Toluene: Ethyl Acetate: Acetic acid: Methanol (3.0:3.0:0.8:0.2). The developed plates were visualized under short UV, long UV, under white light and aderivatised with vanillin sulphuric acid spraying reagent, scanned under UV 254nm, 366nm,540nm (white light) and 620nm (Following derivatisation). Following this, Rf, colour of the spots and densitometric scan were recorded.

The formulation is prepared based on the Asanadi gana explained under Shodhanadi gana sangraha of Vagbhata Samhita.⁴ Asanadi gana is indicated in svitra, kustha, krimi, pandu, prameha, medodosha and other kaphaja vyadhis. The group of same ingredients is explained under Salasaradi gana in Sushruta Samhita.⁵ The ingredients are Asana (Pterocarpus marsupium), Tinisha (Ougenia dalbergioides), Bhoorja (Betula utilis), Svetavaaha (Terminalia arjuna), Prakirya (Sapindus 0trifoliatus), Khadira (Acacia catechu), Kadara (Acacia suma), Bhandi (Albizia lebbek), Shinshipaa (Dalbergia sisoo), Meshashringi (Gymnema sylvestre), Shinshipaa (Dalbergia sisoo), Meshashringi (Gymnema sylvestre), Sveta Chandana (Santalum album), Rakta Chandana (Pterocarpus santalinus), Daruharidra (Berberis aristata), Tala (Borassus flabellifer), Palaasha (Butea monosperma), Jongaka (Aqualaria agollacha), Shaaka (Tectona grandis),Shaal (Shorea robusta), Kramuka (Areca catechu), Dhava (Anogeissus latifolia), Kalinga (Holarrhena antidysenterica), Chhagakarna (Dipterocarpus alatus), Ashvakarna (Dipterocapus alatus). But due to the unavailability of ingredients, an indigenous formulation is formulated and is being used since many years in tertiary care hospital in both the way. The indigenous formulation used for the study has following ingredients

Physico - Chemical parameters

Refractive Index: The refractive index of freshly prepared Asanadi Kashaya was recorded as 1.33529. Whereas, it was recorded as 1.33579 in case of Preservative added Kashaya.

Specific Gravity: The specific gravity of freshly prepared Asanadi Kashaya was 1.003 and it was 1.0158 in case of Preservative added Kashaya.

Viscosity: The viscosity of freshly prepared Asanadi Kashaya was 1.2201 and it was 1.5119 in case of Preservative added Kashaya.

Total Solids: The percentage of total solids of freshly prepared Asanadi Kashaya was 97.92. Whereas it was 95.07 in case of Preservative added Kashaya.

HPTLC

Rf values and colour of the spots in chromatogram developed in Toluene: Ethyl Acetate: Acetic acid: Water (3.0: 3.0: 0.8: 0.2) for freshly prepared Asanadi Kashaya and Preservative added Ashanadi Kashaya were recorded in table 2. HTLC photo documentation (Fig.1) revealed presence of many phyto constituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation).

Solvent system - Toluene: Ethyl Acetate: Acetic acid: Water (3.0: 3.0: 0.8: 0.2)

Track 1 – Asanadi kashaya freshly prepared – 3µl

Track 2 – Asanadi kashaya Preservtive added – 3µl

Track 3 – Asanadi kashaya freshly prepared – 6µl

Track 4 – Asanadi kashaya Preservative added – 6µl

Under short UV (254nm): 7 spots were detected in freshly prepared Asanadi Kashaya (Rf 0.21, 0.30, 0.52, 0.63, 0.67, 0.72, 0.82); Rf 0.52 has dark green colour and all other have light green colour. Whereas, 9 spots were detected in Ashanadi Kashaya with preservative (Rf 0.15, 0.21, 0.22, 0.27, 0.33, 0.52, 0.67, 0.72, 0.80, 0.88) where Rf 0.52 has dark green colour and all other has light green colour.

Under long UV (366nm): 7 spots were detected in Asanadi Kwatha freshly prepared (Rf 0.15 (Flourescent dark green);, 0.21(Flourescent blue);, 0.58 (Flourescent green), 0.67, 0.72, 0.80, 0.88 (Flourescent blue). 8 spots were detected in Ashanadi Kwatha with preservative (Rf 0.15(Flourescent dark green), 0.21(Flourescent blue), 0.58 (Fluorescent dark green), 0.67, 0.72, 0.80, 0.88, 0.92 (Flourescent blue).

White light: One spot was detected in both samples. In freshly prepared Asanadi Kashaya it was detected at Rf 0.81 (purple) and in Ashanadi Kashaya with preservative, it was detected at Rf 0.81 (yellow).

After derivatisation: 7 spots with purple colour were detected in both samples. Rf values of both sample were similar which were Rf 0.22, 0.38, 0.45, 0.58, 0.70, 0.79, and 0.82.

HPTLC Densitometric scan

Under short UV (254nm): Total 9 numbers of peaks were detected in both samples. Among them maximum percentage of area were occupied by Rf 0.04 (32.02%), 0.25 (12.16%), 0.62 (31.65%) in freshly prepared Asanadi Kashaya and Rf (0.04 (27.53%), 0.24 (10.52%), 0.61 (32.58%) and 0.91 (17.41%) in Asanadi Kashaya with preservative as shown in fig.2.

Under long UV (366nm): Total 6 numbers of peaks were detected in both samples. Among them maximum percentage of area were occupied by Rf 0.12 (57.78%), 0.79 (18.84%), 0.93 (13.92%) in freshly prepared Asanadi Kashaya and Rf 0.12 (62.04%), 0.92 (18.03%) in Asanadi Kashaya with preservative as shown in fig. 3.

Under 540nm: Total 6 numbers of peaks were detected in both samples. Among them maximum percentage of area were occupied by Rf 0.04 (26.29%), 0.14 (41.76%), 0.97 (20.88%) in freshly prepared Asanadi Kashaya and Rf 0.04 (41.13%), 0.95 (28.00%) in Asanadi Kashaya with preservative as shown in fig. 4.

Under 620nm: Total 6 numbers of peaks were detected in freshly prepared Asanadi Kashaya. Among them maximum percentage of area were occupied by Rf 0.07 (56.35%), 0.90 (18.85%), 0.94. Total 7 numbers of peaks were detected in Asanadi Kwatha with preservative at Rf 0.08 (43.69%), 0.90 (18.53%), 0.95 (11.14%) as shown in fig. 5.

Discussion

Kashaya is being prepared in large scale because people, in this fast moving world, are more convenient with packaged medications than the raw one that requires more time and effort for the preparation. In addition to this, the method of preparation is followed because of unavailability of raw materials throughout a year. So, Kashaya, for more than a decade, is being prepared in a large scale in Pharmaceutical industries and are being preserved by adding approved preservatives to prolong its shelf life. Patients are advised to dilute the kashaya with water before consuming it. Whereas, freshly prepared kashaya is consumed without diluting it with water. Such type of modification in the method of preparation and administration transforms the qualities of the substance and brings about various changes in the inherent attributes of the substance. It provides new quality that was previously not present in that substance or modifies the existing quality.

Among the physico-chemical parameters, the refractive index and specific gravity were found to be similar in both the form of Kashayas. Whereas, the preservative added Kashaya was more viscous than the freshly prepared one. The increased viscosity of the preserved kashaya can be attributed to the presence of preservative (Sodium Benzoate) in the sample. In addition to this, the presence of more total solids was seen in the freshly prepared Kashaya. Regarding HPTLC analysis, the peaks were found to be more prominent in preservative added Kshaya under the short and long UV. The marked peaks of preserved Kashaya than freshly prepared one can be credited to the addition of Sodium benzoate as a preservative in the Kashaya. There might be interaction of active ingredients of drugs in compound formulations that makes it further difficult to understand those increased peaks.

Conclusion

The samples of freshly prepared and Preserved Asanadi kashaya have been standardized. However, marked variation could not be appreciated in physicochemical parameters and HPTLC analysis of both the samples. Further analysis of phytochemical constituents and other physicochemical parameters can further deepen the understanding related to the minute variations in both the samples. In addition to this, the study of variation in the result due to the interaction of active ingredients in compound formulations can offer new insights in to the existing body of Knowledge.