Indian Journal of Traditional Knowledge
Vol 6(2)-April 2007 -pp 292-297

Preparation, method of optimization and physicochemical evaluation of traditional formulation, Triphala Mashi

Yogesh S Biradar, Piush Sharma & K R Khandelwal *

Department of Pharmacognosy, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane,
Pune 411 038, Maharashtra

E-mail: khandelwal_kr53@rediffmail.com

Received 21 July 2005; revised 30 October 2006

Triphala Mashi was mentioned in Bharat Bhaishjya Ratnakar (2522) and Sharangdhar Samhita-uttar khanda. Triphala Mashi is prepared by using muffle furnace and silica crucible. Physical evaluation of Triphala and Triphala Mashi was done by using DSC and chemical profile of Triphala and Mashi was obtained by preliminary phytochemical screening, total organic carbon content, total inorganic content, ascorbic acid content, HPTLC, and IR. DSC thermograms of Triphala and Triphala Mashi, all are reproducible and can be used as a promising tool for the quality control of the process development. Spectroscopic and chromatographic techniques are proved to be useful in obtaining chemical profile of both Triphala and Triphala Mashi. These techniques are also useful in studying qualitative and quantitative differences in inorganic as well as organic chemical constituents, thermal degradation and conversion of chemical constituents.

Keywords: Triphala Mashi, Ayurvedic formulation, Triphala

IPC Int. Cl.8: C01, A61K36/00

Current advancements in drug discovery technology and search for novel chemical diversity have intensified the efforts for exploring leads from the Indian System of Medicine, Ayurveda. Ayurveda offers knowledge about herbal and herbomineral formulations and there is current need for scientific validation utilizing modern techniques. The present study is an attempt in this direction and involves scientific investigations based on traditional claims mentioned in texts. Mashi means any substance, when burnt, gets blackened and forms amorphous black mass Mashi, followed by Colyrium and Bhasma. The study uses Triphala Mashi as prototype traditional formulation. Triphala Mashi is mentioned in classical Ayurveda texts, Bharat Bhaishjya Ratnakar and Sharangdhar Samhita-uttar khanda. Study involves physical, chemical profiling utilizing modern techniques. The study also compares the effect of heat treatment on physical and chemical properties as compared to Triphala formulation. Triphala Mashi is obtained by thermal stress and contain organic as well as bioinorganic constituents and hence utilizes DSC as part of physical profiling. Chemical profiling includes chromatographic, spectroscopic characterization.

 Triphala (=three fruits) is a traditional Ayurvedic herbal formulation consisting of the dried fruits of three medicinal plants Terminalia chebula (Garten.) Retz., Terminalia belerica (Garten.) Roxb. and Phyllanthus emblica Garten also known as three myrobalan1. Triphala has been described in the ancient Ayurvedic text as a Tridoshic Rasayana, an therapeutic agent with balancing and rejuvenating effects on the three humours or constitutional elements in Ayurveda, vata, pitta and kapha. Triphala is used in Ayurvedic medicine in the treatment of a variety of conditions and also forms part of many other Ayurvedic formulations. Conditions for which Triphala is employed include headache, dyspepsia, constipation, liver conditions, ascites and leucorrhoea. It is also used as a blood purifier, to improve the mental facilities and is reported to posses antiinflammatory, analgesic antiarthritic, hypoglycaemic and antiaging properties2-6.

 When any natural product from vegetable or animal source is heated slowly, at lower temperature (below 450C) Mashi (Black ash) is obtained. If heating is continued further at higher temperatures (above 450C), it forms Bhasma (white ash). This indicates Mashi is nothing but an intermediate product Bhasma, where unlike Bhasma, both organic and inorganic types of constituents are present.

 Mashi is dosage form in which bulk of raw material is reduced to a greater extent by application of certain quantum of energy. Due to this treatment hidden chemical constituent become prominent and / or new chemical moieties are formed which are therapeutically active. Also due to thermal degradation or decomposition thermo labile constituent are lost. Thus without application of any costly method for extraction using organic solvents, we can get therapeutic active organic and inorganic chemical constituents in the form of black mass known as Mashi, by simple heat treatment in controlled manner. If the Mashi is heated further, the burning of carbon particles starts and Mashi gradually is converted to Bhasma, which is undesirable here. The black colour indicates high percentage of carbon and oxides. Non-specific odour and charcoal like taste may be attributed to oxides, inorganic elements and carbon7.

 References about Triphala Mashi were found in Bhaisajyaratnavali and Bharat Bhaisjya Ratnakar (Figs 1 and 2). Bhaisajyaratnavali mentions harada, behada and amla in the same proportion the iron pot, cover it and heat it8. After converting it to the Bhasma, keep it into the glass container. Make its cream in honey and apply it daily, it would heal wound.

Bharat Bhaisjya Ratnakar mentions Triphala and silk cloth in earthen pot and makes its Bhasma9. Mix it in sesamum oil and use it as external application for wound healing.

 

Methodology

 All chemicals and solvents used in the experiments were of analytical grade. Triphala was obtained from local market. Triphala is procured from the local pharmacy, which is prepared by mixing 1:1:1 proportion of dried fruit pulp Terminalia chebula (Garten.) Retz., Terminalia belerica (Garten.) Roxb. and Phyllanthus emblica Garten. 10 gm of Triphala powder at a time is used for the preparation of Triphala Mashi. Steps involved in optimization of method of preparation included heating in muffle furnace in closed crucibles and heating from 30C to desired higher temperature by continuously increasing temperature by 10C /min for about 30 min.

 Aqueous and alcoholic extracts of Triphala and Triphala Mashi were prepared in pure dry powder form. Aqueous extracts (1:6) of Triphala and Triphala Mashi were prepared by hot maceration method and methanol extracts (1:6) of both were prepared using soxhlet apparatus. The extracts were filtered and the solvent was removed using rotary evaporator. The extracts were stored in airtight glass bottle in refrigerator. In order to observe the thermal pathway of Triphala and its Mashi form (obtained by heating at 450C and 600C), study of Triphala and Triphala Mashi with reference to thermal stress was carried out on Differential Scanning Calorimeter (DSC)10. Thermal pathway of Gallic acid was used as marker to study degradation of chemical constituents. Pattern and details of transitions (exotherms and endotherms) can serve a fingerprint tool in the evaluation. Thermo grams were obtained using DSC. Mettler Toledo DSC 821 module and DSC Stare software were used. Heating program: Heating range: 30C-550C; Rate of heating: 10C / min; Rate of nitrogen flow: 100 ml / min.

 The DSC of the selected samples was done by using Aluminum crucibles. The system was purged with nitrogen gas to maintain inert atmosphere.

 Chemical profile of Triphala and Mashi was obtained by preliminary phytochemical screening, total organic carbon content, total inorganic content, HPTLC, and IR. Phytochemical evaluation of aqueous and ethanol extracts of Triphala and Triphala Mashi were prepared prepared by dissolving 500 mg extract in 20 ml of solvent and subjected to preliminary phytochemical testing for the detection of major chemical groups11. The organic matter present in the Triphala and Triphala Mashi was digested with excess of potassium dichromate and sulphuric acid and the residual unutilized dichromate is then titrated with ferrous ammonium sulphate. For detection of the inorganic constituents in the Triphala and Mashi they were dissolved in 50% HCL (v/v) and 50% HNO3 (v/v) for an hour or more, then filtered through ash less filter paper. Finally, selective tests were made for detection of the elements. To confirm the presence of gallic acid, plate was developed in a system comprised of Toluene: Acetone: Glacial acetic acid (3:1:2). It was scanned at 254 nM and derivatised using 5 % FeCl3. IR spectras of Triphala, Triphala Mashi, and Gallic acid were obtained in potassium bromide pellet on FT-IR JASCO 5300 and were expressed in terms of wave number (cm-1).

 

Results and discussion

 In Ayurveda, preparation of Triphala Mashi is mentioned by Anter-Dhum padhati method in Bharat Bhaishjya Ratnakar and Sharangdhar Samhita-uttar khand. For scientific study it is necessary to develop method of preparation of Triphala Mashi. Three steps are involved in the optimization of method of preparation, viz. equipment optimization, optimization of heating pattern, and temperature optimization. For the optimization of equipment, following trials were done: heating in open air in the earthen pot, heating in oven in silica crucible and earthen pot, and heating in muffle furnace in silica crucible, earthen pot and steel container. Due to certain limitations like temperature control, feasibility, applicability in large scale of other methods, heating in muffle furnace in closed crucible was selected as method of preparation of Mashi. Heating pattern was optimized by heating the Triphala powder from 30C to desirable higher temperature by continuously increasing temperature at a rate of 10C/min and heating the Triphala at fixed higher temperature for fixed time. Due to the threat of charring of chemical constituents it is practicable to heat the raw material from low temperature to desirable higher temperature.

 Mashia dosage form is obtained as a result of thermal stress. So, temperature optimization is the most important step in the study of Mashi. Determination of optimum temperature, at which it maintains its physical, chemical characteristics, is of prime importance. Triphala turns black at around 450C temperature; hence Mashi preparations at temperatures 450C, 500C, 550C, and 600C were selected for the study. While preparing Triphala Mashi, free flowing powder of Triphala gets converted into the hard mass. This may be attributed to the changes occurring in the physical and chemical properties of the drug due to thermal stress. During extracts preparation more amount of frothing was observed in Triphala aqueous extract than aqueous extract of Triphala Mashi, alcoholic extract of Triphala and Triphala Mashi, which may be due to presence of more saponins in aqueous extract of Triphala than Triphala Mashi. Colour of different extracts of Mashi was blackish indicating presence of organic carbon in the Mashi form. Difference in the extractive values of Triphala at 450C and 600C Mashi indicates that there was more chemical degradation in 600C Mashi. There was temperature dependent degradation of chemical constituents (Table 1).

 

Table 1Yield % of Triphala Mashi, aqueous extractive value, alcoholic extractive value

 

 

 

 

Sample

Yield % of Triphala Mashi (%w/w)

Aqueous extractive value (%w/w)

Alcoholic extractive value (%w/w)

 

 

 

 

Triphala

 

44.76

33.7

450C Mashi

77

13.16

6.04

600C Mashi

47

0.08

0.03

 All the four samples showed first endothermic peak below 150C indicating loss of moisture content or volatile matter. All the four samples were having totally different DSC pattern (Figs 3-6). In Triphala, second peak was observed between 161.09C-212.31C and third peak between 286.58C-339.53C. These two peaks were not observed in the 450C and 600C Mashi because these constituents may be lost during formation of Mashi due to thermal stress. Degradation pattern above 350C is not prominent in Triphala but it was seen in Triphala Mashi because there may be transformation of constituents in this temperature range during formation of Triphala Mashi. DSC pattern of gallic acid was taken as marker to study the degradation of the gallic acid in Triphala Mashi. There was no matching of gallic acid peak with other samples that means there is no degradation of gallic acid when it is present in complex form with other constituents. DSC pattern of gallic acid showed sharp endothermic peak between 266.78C and 273.73C, where degradation / decomposition takes place but such transition couldnt be seen in any other sample like Triphala, Triphala Mashi samples prepared at 450C and 600C indicating that degradation / decomposition of gallic acid is not taking place in those samples. This protection may be due to its chemical complex in which it is present in these samples. Further study is required for exploring the correct answer to this. Chromatographic study also supports to the conclusion that decomposition / degradation of gallic acid doesnt take place up to a temperature 450C.

 Total organic carbon content of Triphala, 450C and 600C Mashi was 50.005 %, 47.792 %, 2.443 %, respectively (Table 3). Inorganic content is deter-mined by AAS, and it was observed that there is increase in (% w/w) content of inorganic elements as the temperature increases. This was due to loss of some organic matter from the formulation due to their thermal degradation. Potassium is the major inorganic element present in the Triphala Mashi form (Table 4). Study reveals that degradation/decomposition of anthraquinone takes place as temperature increases while phenolic compounds like tannins; flavonoids and ascorbic acid remain stable even at higher temp (Table 2). Ascorbic acid content for Triphala, 450C and 600C Mashi were 0.3337, 0.3187 and 0.04485, respectively (Table 7). Using gallic acid as marker HPTLC analysis of Triphala and Triphala Mashi was done (Fig. 7). Mobile phase Toluene: acetone: Glacial acetic acid (3:1:2) was used for the study and sprayed with 5 % Fecl3. In this system, alcoholic extracts of Triphala and 450C Mashi were compared with the marketed Triphala for gallic acid. All the samples showed sharp peaks at Rf value 0.44 like authentic sample of gallic acid. This confirms that gallic acid remains present at 450C.

Table 2¾Preliminary phytochemical screening

 

 

 

 

 

Chemical Test

Triphala aqueous

Triphala .Mashi aqueous

Triphala

alcoholic

Triphala Mashi

alcoholic

 

 

 

 

 

Alkloids – Dragendorff’s reagent test

-

-

-

-

Anthraquinone glycosides – Bornatrager test

+

-

+

-

Saponins – Foam test

++

+

-

-

Steroids and Triterpenoids

-

-

-

-

Phenolic compounds Tannins

++

++

++

++

Proteins

-

-

-

-

Ascorbic acid

+

+

+

+

 

 

 

 

 

 

 

 

Table 3Total organic carbon content

 

 

Sample

Total Organic Carbon (% w/w)

 

 

Triphala

50.005

450 0 C Mashi

47.792

600 0 C Mashi

2.443

 

 

Table 4Inorganic content

 

 

 

 

Inorganic element

Triphala (%w/w)

450C Mashi (%w/w)

Method

 

 

 

 

Calcium

0.416

0.5937

AAS

Magnesium

0.381

0.5428

AAS

Potassium

1.56

2.2276

Flame photometer

Zinc

0.0027

0.00398

AAS

Iron

0.049

0.07234

AAS

Phosphates

0.231

O.33

Visible spectrophotometer (690 nm)

Sodium

0.035

0.05452

Flame photometre

Sulphates

0.105

0.0759

Visible spectrophotometer (420 nm)

Chlorides

0126

0.1835

Volumetry

Hydroxides

Nil

Nil

Volumetry

Lead

Nil

Nil

AAS

Aresnic

Nil

Nil

AAS

Mercury

Nil

Nil

AAS

 

 

Table 5Ascorbic acid determination

 

 

Sample

Ascorbic acid content

(% w/w)

 

 

Triphala

0.3337

4500 C Mashi

0.3187

6000 C Mashi

0.04485

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 IR spectral of Triphala and Triphala Mashi were taken to study functional group degradation due to thermal stress (Fig. 8). All the three constituents of Triphala churna known to contain gallic acid and therefore IR spectra of gallic acid along with the spectra of Triphala, Triphala Mashi and gallic acid heated at 450C were recorded to study gallic acid degradation. The IR spectra of gallic acid showed typical frequencies due to -OH stretching vibrations of both phenolic as well as hydroxyl group of carboxylic acid and carbonyl stretching frequencies12. Therefore, the bands in the region 3390-3200 cm-1 can be assigned to -OH stretching vibrations. The bands in the region 1725-1600 cm-1 may be assigned to C=O stretching vibrations. All these bands were also observed in Triphala, 450C Mashi, 600C Mashi with slight shifting of bands while these bands were not observed in 700C Mashi and gallic acid heated up to 450C. In all the spectra, there was a band of low intensity at 2360 cm-1, which could not be assigned to any of the frequencies attributable to gallic acid. One more interesting feature observed was the presence of the band of variable intensity in the region of 1250-1200 cm-1in gallic acid and Triphala and absent in thermally treated samples. From the IR spectra of Triphala churna, its Mashi form, gallic acid and gallic acid heated up to 450C it is observed that gallic acid is also present in its Mashi form of Triphala. However, if pure gallic acid is heated to the same temperature it is degraded, it showed that gallic

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 acid is protected from degradation if it is present in complex with other constituents.

 

Conclusion

 After evaluating the different samples of Mashi as a whole and their extracts, from the above results 450C was regarded as the optimum temperature for the preparation of Triphala Mashi. Further study is necessary to explore and justify the biological claims of Triphala Mashi, which are mentioned in Ayurvedic texts. Also during TLC studies, some new bands were observed in Triphala Mashi, which were not present in Triphala that may be due to reaction due to heat treatment. So, further fractionation and studying biological effect is necessary to get new lead molecule from Triphala Mashi. There is need to pursue the characterization of active principles, to optimize the observed activities.

 

Acknowledgement

 Authors are thankful to AICTE for the fellowship to Mr Yogesh S Biradar. The generous help of Accurate Analytical Laboratory, Pune-53 (FDA Lic.No. PD/TL-9 dated 13.10.1997) by way of extending the laboratory facilities is gratefully acknowledged.


References

1         Hans Wohlmuth, Triphala a short review, Information Res Bot Med, 16, 2.

2         Jagetia GC, Baliga MS, Malagi KJ & Kamath M, The evaluation of the radioprotective effect of Triphala (an Ayurvedic rejuvenating drug) in the mice exposed to radiation, Phytomedicine, 9 (2) (2002) 99.

3         Rege NN, Thatte UM & Dahanukar SA, Adaptogenic properties of six rasayana herbs used in Ayurvedic medicine, Phytother Res, 13 (4) (1999) 275.

4         Mehta BK, Shitut S & Wankhade H, In vitro antimicrobial efficacy of Triphala, Fitoterapia, 64 (4) (1993) 371.

5         Vani T, Rajani M, Sarkar S & Shishoo CJ, Antioxidant properties of the Ayurvedic formulation Triphala and its constituents, Int J Pharmacog, 35 (5) (1997) 313.

6         Kaur S, Arora S, Kaur K & Kumar S, In vitro antimutagenic activity of Triphala - an Indian herbal drug, Food Chem Toxicol, 40 (4) (2002) 527.

7         Ambike AA, Khandelwal KR & Jadhav BK, Mashi, an Ayurvedic dosage form, Deerghayu Int, 76, 19(4) 239.

8         Bhaisajyaratnavali Sloka No 52 (8) (Chowkhmba Sanskrit Sansthan, Varanasi), 611.

9         Bharat Bhaisjya Ratnakar Sloka No 2522 and 2523, (B Jain Publishers Private Limited), 389.

10      Sharma BK, Instrumental Methods of Chemical Analysis, (Krishna Prakashan, Meerut), 1998, 247.

11      Khandelwal KR , Practical Pharmacognosy, (Nirali Prakashan, Pune ), 2000, 149.

12      Bahulikar AS, Kashalkar RV, Pundlik MD, Infrared spectrometry in studies on herbal drug Triphala churna, Asian J Chem., 15 (2) (2003) 851.


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