Abstract
Basti therapy is used widely for treatment of various diseases in the field of Ayurveda with several proven clinical applications, however; the exact mechanism through which the benefits are obtained are yet to be ascertained in the light of the contemporary developments in clinical science. It is also not clear that when administered Basti the active principles in the formulation gain entry in to the systemic circulation or not. The present study was planned to acquire some preliminary data with regards to the absorption of phytochemical constituents of the formulations when administered in the form of Basti. Gallic acid was used as marker compound and it's absorption in systemic circulation was assessed using high performance liquid chromatography. Gallic acid present in Lekhana Basti (Emaciating/ Desiccating Medicated Enema) was found to get absorbed in to systemic circulation. Maximum concentration in serum was seen in the animal which received Lekhana Basti with Prakshepa Dravya ( Catalytic Adjuvant) in comparison to animal which received Lekhana Basti without Prakshepa Dravya. Area under curve in chromatogram was also more in animal which received Lekhana Basti with Prakshepa Dravya. From primary evidence created by HPLC study it can be said that, phytochemicals of the Basti formulation do get absorbed in systemic circulation.
Keywords: Lekhana Basti, Prakshepa Dravya, pharmacokinetic, Gallic acid, albino rats
Introduction
Panchakarma therapies are popular in the field of Ayurvedic disease management. Though their effect and safety is well established by the evidence of experiences since many centuries, their mode of action in front of contemporary scientific point of view is still a question. To take Ayurveda to the global platform it is necessary to explain the complete pharmacokinetics of Ayurvedic medicines and mode of actions of available therapies. Basti being the most widely used and highly effective treatment modality in the Ayurveda, it is the prime subject of interest for modern scientific community. With this background the basic question which comes forward regarding Basti is, “do active principles of drugs used in Basti get absorbed in systemic circulation?” To find an answer to this question an animal experiment was undertaken to ascertain whether a marker compound gains entry in to the systemic circulation when administered by Basti and the influence of other factors on this absorption.
Basti is a multidrug formulation that is given per rectum and reaches up to ileo-caecal junction as seen in previous studies1. Lekhana Basti (Emaciating/Desiccating Medicated Enema) used in present study has Triphala Kashaya (Decoction) as a main ingredient. Triphala is a composition of Haritaki (Terminalia chebula Retz.), Bibhitaka (Terminalia belerica Roxb.) and Amalaki (Emblica officinalis Gaertn.) fruits in equal quantity. Gallic acid is a marker compound for Triphala2. In the present study absorption of gallic acid present in Basti was assessed.
Materials and Methods
Test formulation
The raw materials Table-1 of Lekhana Basti3 were procured from pharmacy attached to Gujarat Ayurved University, Jamnagar and authenticated by pharmacognosist. Triphaladya Taila4 was used as Sneha(oil) & Putoyavanyadi Kalka5 (Paste of hurbs) as it is a commonly used Kalka for Basti. From these ingredients Lekhana Basti was prepared by classical method6, freshly every day just before administration.
Table No. 1.
Contents of the Lekhana Basti (For preparation of 12 Prasruta i.e. approximately 1000ml of Niruha)
Animals
Male Wistar strain albino rats weighing 280g were obtained from animal house attached to the Pharmacology Laboratory. Animals were exposed to natural day and night cycles with ideal laboratory condition in terms of ambient temperature (22 ± 2°C) and humidity (50 - 60%). They were fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water given ad libitum. All procedures and experiments were conducted in day time according to specification of the Indian National Science Academy (INSA). The experiments were carried out after obtaining the permission of Institutional Animal Ethics Committee (Approval number; IAEC/07/2010/09/MD).
Dose fixation
The quantities of ingredients in liquid form were determined in ml by considering the specific gravities of the basic component of the formulation and converting the grams to ml. Thus, the final prepared 12 Prasruta (Which is the ideal dose of an adult as per Ayurvedic classics7) Basti comes to be of 1000 ml. For present trial half of the standard dose i.e. Ardhamatrika Basti was considered as the adult human dose i.e. 500ml for Niruha which is used now a days practically. The dose of Lekhana Basti was calculated by extrapolating the therapeutic dose to rat dose on the basis of body surface area ratio by referring to the table of Paget and Barnes (1964)8 and by calculating this way, the rat dose for Niruha Basti is 45ml/kg.
Prior to the experiment proper, to rule out any possible practical difficulty while administering this particular dose of Niruha Basti (Decoction Enema)and to ascertain up to what region of colon this dose of Basti will reach, a pilot study was carried out in which two rats were used. First rat was administered with above mentioned dose of Niruha Basti and observed for any ill effect. Further the animal was sacrificed about 5 minutes after the Basti administration with over dose of ether anaesthesia and its intestinal tract was observed for presence of Basti material. The Basti contents were found to reach up to caecum of the animal. Second animal was first sacrificed with over dose of ether anaesthesia and the abdomen was opened by midline incision and the large intestine was carefully exposed. Then the Niruha Basti was given through rectum. It was confirmed that it reaches up to the ileocaecal junction. Thus the dose decided for the animals was fixed for the present study.
Administration of the test drug
Lekhana Basti was administered to the overnight fasted animal after doing local Abhyanga (Oleation) at lower abdomen and lower back with luke warm Triphaladya Taila (medicated oil)and Swedana (Sudation) with hot water filled in a polythene bag. To administer the test drug, simple rubber catheter (no. 4) attached to 20cc plastic syringe was used.
Experimental protocol
The selected rats were named as Rat ‘A’ and Rat ‘B’. Rat ‘A’ was given Lekhana Basti with Prakshepa (Catalytic Adjuvant) and Rat ‘B’ was given Lekhana Basti without Prakshepa to find some incidental difference that might be possible by addition of Prakshepa this protocol of test to animals was selected.
The selected animals were fasted over night and blood sample of both animals were collected. Then Lekhana Basti as per fixed dose was administered to respective animals and subsequent blood samples were collected after Basti at different time intervals (i.e., after 45min, 90 min and 180min of Basti administration). From the blood samples serum was separated by centrifuging at 3000 rpm for 10 minutes. The serum samples were subjected to HPLC analysis for the presence of gallic acid or other derivatives.
High performance liquid chromatography (HPLC) analysis
Chromatographic system
High performance liquid chromatographic system equipped with LC-20AD pump, SPD- M 20 AD 10 AS vp photo array detector in combination with LC 10A software was used.zw
Chromatographic conditions
Mobile phase: Water : Methanol : Acetic acid (85 : 15 : 5)
Column: Inertsil C18 250 mm × 4.6 mm i.d. with a 5 μm particle size.
Detector: SPD- M 20 AD 10 AS vp photo array detector
Wavelength for recording chromatogram: 254nm
Flow rate: 0.8ml/min
Inject volume: 20μl
Standard preparation
50mg of Gallic acid Reference standard (Loba chemie-99.5%) was weighed accurately and taken in to a 50 ml volumetric flask. Dissolved and made up the volume with methanol.
Sample preparation
500μl serum was adjusted to pH 2.5 with 150μl of 1 mol potassium dihydrogen phosphate solution and 15μl phosphoric acid.
2.5ml of acetonitrile was added for 1minute and centrifuged at 3500 rpm for 10min.
Supernatant liquid was evaporated to dryness at 35°C.
Residue reconstituted in 2 m l methanol.
The solution was filtered through Whatman filter paper (No. 41) in a dry stoppered tube. This solution was used for HPLC analysis.
Procedure and data analysis
The Chromatographic System was set up as above. Equal volumes (about 20μl) of the standard and the sample were separately injected into the chromatographic system. The chromatograph for qualitative percentage of Gallic acid was recorded and any peaks from the mobile phase preparation were discarded. Quantitative determination was carried out with previously used external standard method9 using microsoft excel sheet. Maximum serum concentration was determined by visual inspection of the data. Area under curve (AUC) was calculated using trapezoidal rule using values obtained up to 90minute samples.
Results
Gallic acid standard was found to have retention time of 2.720 min as seen in Figure-1 which shows the HPLC chromatogram of standard Gallic acid. Figure-2 shows the absence of Gallic acid in the serum collected before Basti whereas Fig 3 & 4 shows the presence of Gallic acid.
Fig. 1.
HPLC chromatogram of Standard Gallic acid
Fig. 2.
HPLC chromatogram of serum of Rat A before Basti administration.
Fig. 3.
HPLC chromatogram of serum of Rat A after 90 minutes of Basti administration.
Fig. 4.
HPLC chromatogram of serum of Rat B after 45 minutes of Basti administration.
The details of concentration were as depicted inTable-2, which shows that, the Gallic acid was not detected in the blood sample taken before Basti.
Table No.2-.
Concentration and retention time of Gallic acid in Serum
However, the compound was detected in the samples taken after Basti in both the animals. The concentration was found maximum after 90 min of Basti in Rat ‘A’ i.e. 112.96 μmol/L which found to be reduced to 58.07 μmol/L in sample taken after 180 min. In Rat ‘B’ the concentration was more i.e. 65.98 μmol/L in sample taken after 45min, followed by 48.42 μmol/L in 90min sample.
Figure- 5 shows serum concentration-time profile of Gallic acid after Lekhana Basti administration in Rat A and Rat B.
Fig. 5.
Serum concentration-time profile of Gallic acid after Lekhana Basti administration in Rat A and Rat B
The details drawn from this curve stating the pharmacokinetic aspect of Lekhana Basti are as shown in table-3.
Table no. 3-.
Pharmacokinetic parameters of Gallic acid after single Lekhana Basti administration
C 2 μmol/L: Maximum serum concentration
tmax min: Maximum time
AUC(0-90min.) μmol.min./L: Area under Serum concentration time curve
Discussion
The efficacy of Basti is believed to be due to its action by the Virya10(Active Principles). Bastivirya gets spread all over the body through Srotasa11(Minute channels of the body). Virya by definition is any of the Guna(Property) among Vimshati (Twenty) Guna12. As Guna are Dravyashrita13(Resides in Drug); to prove the absorption of the Bastivirya, absorption of some phytochemical that can be linked to the specific Guna is necessary. Hence for the present study Gallic acid was selected as marker compound to ascertain whether drugs present in Basti Dravya will get absorbed or not.
The absorption of phytochemical constituent of Basti formulation i.e. Gallic acid in present study has supported the theory of action of ‘Bastikarma’ through ‘Bastivirya’ which states that, when Basti is administered in the Pakvashaya (chiefly Colon), its Virya is taken up by Samana Vayu with the help of Apana Vayu. Then it reaches other types Vayu also and affects them by its action. It also keeps Pitta and Kapha in their proper places. The transport of Bastivirya is by Kedarikulya Nyaya which makes it spread all over the body by virtue of different Vayu14.
Maximum concentration Gallic acid was seen in Rat A (112.96 μmol/L) though Rat B serum has achieved maximum concentration early (45min). Area under curve was more in Chromatogram of Rat A (4319.56 μmol.min./L) than that of Rat B (4058.55 μmol.min./L). This shows that absorption of Gallic acid was more in Rat A as compared to Rat B. This may be due to effect of Prakshepa in the Lekhana Basti which has mild irritant properties causing mild inflammation to colon, this inflammation may influence the drug absorption as follows15-
Drug may be absorbed more as a result of general increase in capillary permeability due to intestinal inflammation.
Inflammation may decrease the multidrug resistance function in the intestinal wall epithelium, resulting in decreased pumping of drug from the enterocyte into the intestinal lumen.
Gallic acid is a marker compound for Triphala (A combination of 3 drugs). As absorption of Gallic acid is confirmed by HPLC it provides a primary evidence for absorption of phytochemical constituents of the Basti drug in the systemic circulation, the probable site of absorption being colon, which can be further evaluated.
From the results obtained it becomes clear that the active principles in the formulation get absorbed probably through different parts of the GI tract from rectum up to ileocecal junction. The presence of adjuvants in the form of Prakshepa Dravyas seems to influence the absorption- the exact nature of the influence remains to be determined. It is necessary to undertake detailed studies using more number of animals (8-10 in a group) so that the pharmacokinetic profile can be determined in greater detail by including the entire phytochemical spectrum of Triphala combination. Once this is done then the influence of different factors like time of Basti, rate at which it is given, role of adjuvants etc., can be studied in greater detail so that optimum conditions to obtain better therapeutic benefits can be defined. Based on the data obtained from such studies the same can be extended to human beings. This can be considered as the first step in developing standard operative procedures for different types of Basti formulations.
Conclusion
From primary evidence obtained by HPLC study it can be said that, phytochemicals of the Basti formulation do get absorbed in systemic circulation and the concentration and rate of absorption is dependent upon properties of its constituents like Prakshepa etc. The study provides first direct evidence for the absorption of active principles of Triphala through Basti and indicates the necessity of undertaking detailed pharmacokinetic study to optimise the therapy so that its therapeutic application can be refined. By extending the results to human beings it may be possible to determine the Standard Operation procedure (SOP) for different kinds of Basti formulations.
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