Physio-chemical properties and Antimicrobial Activity of Triphala Masi
Dr. Riya Sinha*, Dr. Jeevesh K.B.
Department of Rasashastra & Bhaishajya Kalpana, Ramakrishna Ayurvedic Medical College, Hospital and Research Center, Bengaluru-560064, Karnataka, India
Received: 03-Jul-2018 , Accepted: 07-Oct-2018
Keywords: Triphala Masi, Antimicrobial Activity, Physico-chemical analysis
How To Cite
Triphala is a traditional Ayurvedic herbal formulation consisting of the dried fruits of three medicinal plants Terminalia chebula, Terminalia belerica and Phyllanthus embelica. Triphalais also main ingredient of Triphala Masi and practiced very less which is unexplored. In consideration of all above the present study was planned to make an attempt to evaluate the pharmaceutical analysis and evaluation of antimicrobial activity of Triphala Masi. The Triphala Masi was prepared by heating at 450 °C for 150 min. The physical values like total ash, acid insoluble ash, water-soluble ash, loss on drying, pH value and loss on drying were determined. The organoleptic characters of prepared Triphala Masi were studied. The prepared Masi were black colour with charcoal like taste and burnt smell. Total ash, Acid insoluble ash, water soluble ash and loss on drying of Triphala Masi fulfill the standards criteria. pH value suggested to be acidic for Triphala Masi. Triphala Masi exhibited a broad-spectrum antimicrobial activity and inhibited the growth of S. aureus and E. coli. The above findings of pharmaceutical, analytical profile and antimicrobial study of Triphala Masi indicates that the formulation was stable and has effective antibacterial activity.
Presently Ayurveda is consisting ofhuge number of formulations to accomplish its objectives. Masiis one of such preparation and employed for controlled of various diseases. Masi is a solid dosage form in which bulk of raw material is reduced to a greater extent by application of a certain quantum of energy. As a result of this treatment hidden chemical constituents become prominent and/or new chemical moieties are formed which are therapeutically active. Due to thermal degradation or decomposition thermo labile constituent are lost. Therapeutically active organic and inorganic chemical constituents can be prepared by simple heat treatment in a controlled manner. The black color indicates high percentage of carbon and oxides. Non-specific odor and charcoal like taste may be attributed to oxides, inorganic elements and carbon1-4.
The antibiotics available for the bacterial infection have developed resistance against bacteria. The novel drug delivery system or development of novel formulation is best option to overcome this problem. Masi Kalpana is an important pharmaceutical preparation mentioned in Ayurveda Pharmaceutics. Masi is a dosage form producing higher extent of therapeutic effect compared to other Ayurvedic dosage form due to application of a certain quantum of energy. As a result of this treatment hidden chemical constituents become prominent and/or new chemical moieties are formed which are therapeutically active4,5.
Triphalais a traditional Ayurvedic herbal formulation consisting of Terminalia chebula, Terminalia bellerica and Emblica officinalis. Triphala is used in Ayurvedic medicine to treat a variety of conditions like headache, dyspepsia, blood purifier, constipation, liver conditions, ascites, skin disease, intermittent fever, leucorrhoea etc.
Triphala in Ayurvedic system of medicines widely used in different dosage forms in different conditions. Among them Kashaya it is used for vrana prkshalana, netra prakshalana etc indicating about its bactericidal action6-8.
Triphalais also main ingredient of Triphala Masi and practiced very less which is unexplored. In consideration of all above the present study was planned to make an attempt to evaluate the pharmaceutical analysis and evaluation of antimicrobial activity of Triphala masi.
2 Materials and Methods
2.1 Collection of the drug
The raw drugs required for the preparation of medicine was procured from locality of Bangalore, Karnataka.
2.2 Authentication of the drug
The authentication of the raw drug was done at the Rasashastra and Bhaishajya Kalpana P.G. department laboratory, Ramakrishna Ayurvedic Medical Collage, Hospital and Research Center, Yalahanka, Bangalore.
2.3 Preparation of Triphala Masi
The Triphala Masiwas prepared at Rasashastra and Bhaishajya Kalpana P.G. department laboratory, Ramakrishna Ayurvedic Medical Collage, Hospital and Research Center, Yalahanka, Bangalore.The Triphala Masiwas prepared with classic reference (Table 1). The plant material used in preparation of Triphala Masi demonstrated in table 1.
Place of pre-processing of the drug like cleaning, drying was done at Rasashastra and Bhaishajya Kalpana P.G. department laboratory, Ramakrishna Ayurvedic Medical Collage, Hospital and Research Center, Yalahanka, Bangalore.
The dried fruits of Terminalia chebula, Terminalia bellerica and Emblica officinalis were taken and made into powder called as churn. Further 150 gm churn of Terminalia chebula, Terminalia bellerica and Emblica officinalis individually weighed and were mixed to form Triphala churn. The Triphala was heated in closed silica crucibles in a muffle furnace. Triphala was heated from 30 °C to 450 °C higher temperature by continuously increasing temperature to 10 °C/min. Then it was filtered with cloth and preserved in the glass containers9, 10.
2.4 Analytical study
2.4.1 Organoleptic parameters11,12
Organoleptic (i. e., impression on the organs) refers to evaluation by means of the organs of sense and the odor, taste etc. of the drugs. The macroscopic characteristics of the drug include:
2.4.2 Physico-chemical analysis13-20
The physical values like total ash, acid insoluble ash, water-soluble ash, loss on drying, pH value and TLC were determined.
Determination of total ash value
Accurately weighed about 3 gms of air dried powdered drug was taken in a tared silica crucible and incinerated by gradually increasing the temperature to make it dull red hot until free from carbon. Cooled and weighed, repeated for constant value. Then the percentage of total ash was calculated with reference to the air dried drug.
Determination of acid insoluble ash value
The ash obtained as directed under total ash was boiled with 25 ml of 2N HCl for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water, ignited and weighed, then calculated the percentage of acid insoluble ash with reference to the air dried drug.
Determination of water soluble ash value
The total ash obtained was boiled with 25 ml. of water for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited for 15 minutes at a temperature not exceeding 450°C. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represents the water soluble ash. The percentage of water soluble ash was calculated with reference to the air dried drug.
About 1.5 gm of powdered drug was weighed accurately in a tared porcelein dish which was previously dried at 105 °C in hot air oven to constant weight and then weighed. From the difference in weight, the percentage loss of drying with reference to the air dried substance was calculated.
Determination of pH
1 ml of sample was taken and mark up to 10 ml with distilled water, stirred well and filtered. The filtrate was used for the experiment. Instrument was switched on. 30 minutes time was given for warming pH meter. The pH 4 solution was first introduced and the pH adjusted by using the knob to 4.02 for room temperature 30 °C. The pH 7 solution was introduced and the pH meter adjusted to 7 by using the knob. Introduced the pH 9.2 solution and checked the pH reading without adjusting the knob. Then the sample solution was introduced and reading was noted. Repeated the test four times and the average reading were taken as result.
2.5 Preparation of aqueous extract of Triphala Masi
The powder of Triphala Masi packed well in soxhlet apparatus and extracted with distilled water until the completion of the extraction. The extract was filtered while hot, and the solvents were removed by distillation and the last traces of solvent being removed under reduced pressure. The aqueous extracts were stored in refrigerator for further experimental work.
Qualitative chemical tests were performed to determine the presence of alkaloids, carbohydrates, cardiac glycosides, polyphenols, saponins, tannins and terpenoids.
2.6 Thin layer chromatography (TLC)21-26
The drug sample was prepared with the respective solvent ethanol and distilled water and made up to 10 ml in different test tubes. Then the samples were taken in a capillary tube and it was spotted in preparative TLC plates coated with silica gel G. The plates were developed in TLC chamber previously saturated with different solvent systems. The different spots developed in each solvent system were identified by means of detecting agent and the Rf value are correspondingly calculated.
2.7 Anti-microbial study
Theaqueous extracts of Triphala Masi was prepared and stored in refrigerator for further experimental work.
2.7.1 Test microorganisms
Pure chemical isolates of Staphylococcus aureus and Escherichia coli were used for studies.
2.7.2 Culture medium and inoculum preparation
High sensitivity testing agar (Hi-Media) was used for checking antimicrobial activity of extract against S. aureus and E. coli. The microbial strains were cultured on the slants in the sterilized Laminar Air Flow from the pure culture. These cultured slants were incubated at 37 °C for bacterial growth for 2–3 days. High sensitivity testing agar was mixed at a concentration of 23.4 g/1000 ml in distilled water and autoclaved at 121 °C for 15 min. A loop full from pure culture of a bacterial strain was mixed in the 10 ml of Nutrient broth medium and incubated at 37 °C overnight and the activated culture was used for streaking onto the agar plates for antimicrobial sensitivity.
2.7.3 Agar well diffusion assay
The antibacterial activity of the aqueous extract of Triphala Masi at different concentration (100 µg/ml and 200 µg/ml) was determined by Agar well diffusion assay. The standard drug Ciprofloxacin (25 µg/ml) was used to evaluate antibacterial activity. 2.34 gm of high sensitivity testing agar was dissolved in 100 ml of distilled water and autoclaved at 121 °C for 15 min. Before transferring this medium in sterilized petri plates, it was allowed to cool and then was poured into the petri plates and allowed to solidify.
After this, it was inoculated with activated culture using sterile cotton swabs. And the wells were created using sterile agar borer and the wells were filled by adding 25 μl of extract using micropipette and were incubated at 37 °C for 12–24 h. Three replicates were carried out for extract against the test organisms. After incubation, the diameters of the zones of inhibition (ZOI) were measured in millimeters, and the mean values were tabulated27-31.
2.7.8 Minimum inhibitory concentration (MIC)
The extracts, which showed antibacterial activity in the agar well diffusion assay, were subjected to the MIC assay. Serial two-fold dilutions of the extracts were prepared in nutrient broth with different concentrations (ranging from 200 to 0.39 and 100 to 0.39 mg/ml). An equal volume of inoculum (1 ml) was added in all the tubes. The tubes (total volume 2 ml) were incubated at 37 °C for 18 h. The MIC values were interpreted as the highest dilution (lowest concentration) of the sample, which showed clear fluid with no development of turbidity. Solvent blanks and positive controls were also included. All tests were performed in triplicate30, 31.
2.8 Statistical analysis
Values are mean ± SEM of three replicates in results for physicochemical analysis.
Results were analyzed using one way analysis of variance (ANOVA) followed by the Dunnett`s test by using statistical software package, Graph Pad Prism; version 7 for antibacterial activity.
3 Results and Discussions
3.1 Triphala Masi
Table 2 demonstrated that about 6% drug loss during powder of plant materials.
Table 3 shows that there was drug loss about 13.6 during preparation of Triphala Masi.
The colour of Triphala Masi was changes on heating the Triphala at various temperatures (Fig 1).
Triphala Masi was obtained, when Triphala Churn was heated slowly, at lower temperature (generally below 450 °C). If heating is continued further at higher temperatures (above 450 °C) it forms Bhasma (White ash).
Mashi is an intermediate product of Bhasma in which unlike Bhasma, both organic and inorganic constituents are present.
For preparation of Triphala Masi sample in muffle furnace, 150 minutes were required and temperature given was 400 °C. Minimum six hours were required for self cooling.
Masi is a dosage form in which bulk of raw material is reduced to a greater extent by application of a certain quantum of energy. As a result of this treatment hidden chemical constituents become prominent and/or new chemical moieties are formed which are therapeutically active.
Due to thermal degradation or decomposition thermo labile constituent are lost. Therapeutically active organic and inorganic chemical constituents can be prepared by simple heat treatment in a controlled manner. The black color indicates high percentage of carbon and oxides. Non-specific odor and charcoal like taste may be attributed to oxides, inorganic elements and carbon.
3.2 Organoleptic properties
Table 4 shows the black colour of Masi with charcoal like taste and burnt smell products.
Weight of Triphala Masi - 361.4 gms
Weight loss - 13.6 gms
3.3 Analytical study
3.3.1 Total Ash
It is the criteria for identity or purity of drugs. Total ash is inclusive of physiological ash derived from plant tissue and non physiological ash consists of residue of the extraneous matter such as sand, soil etc. adhering to the herb itself. Total Ash value of Triphala Masi was 3.4±0.62% w/w (Table 5). The total ash value of Triphala Masi was low in number which refers the less amount of non physiological Ash refers to purity of the drug.
3.3.2 Ash value
Water soluble Ash and acid insoluble ash also has been shown less value for of Triphala Masi (0.46±0.06% w/w and 1.7±0.36% w/w) (Table 5). Less acid insoluble ash value refers less adherent dirt and sand particles. This can be used as criteria for quality control purpose.
3.3.3 Loss on drying
Triphala Masi (0.87±0.04% w/w) have lower value of loss on drying (Table 5). This test is to detect the moisture and volatile content in the sample. Due to low loss on drying value, formulation might less susceptible to microbial contamination.
Mean pH value of Triphala Masi was shown in acidic nature (Table 6). It was facilitated the absorption of drug at the stomach level. The results obtained in pH were found under the limits of Ayurvedic Pharmacopoeia of India.
3.4 Phytochemical screening
Preliminary phytochemical investigations of the extracts of Triphala Masi revealed the absence of chemical constituents (Table 7). The findings confirm that the chemical constituents are thermolabile and lost during heat treatment for preparation of Triphala Masi.
TLC photo documentation of Triphala Masi explains the presence of total 1 spots also the Rf value – 0.71 (Table 8). Suggested the presence of flavonoids in the sample which confirms the genuenity of drug.
3.6 Antibacterial activity
The diameter of the inhibition of zone of Triphala Masi on gram positive at different concentrations, by disc diffusion method, was determined to access its antibacterial effect.
The Triphala Masi at the dose of 100 mg/ml and 200 mg/ml demonstrated 4.3±0.85 and 11.7±1.34 mm ZOI for S. aureus, respectively. The ciprofloxacin exhibited zone of inhibition of 14.8±1.35 mm for S. aureus (Table 9).
The MIC value for Triphala Masi 100 mg/ml, Triphala Masi 200 mg/ml and Ciprofloxacin were 6.25, 3.125 and 1.562 for S. aureus, respectively (Table 10).
In general, the aqueous extracts of Triphala Masi exhibited a broad-spectrum antimicrobial activity against gram positive and gram negative. It inhibited the growth of S. aureus and E. coli. In agar diffusion method, the extract inhibited the growth of S. aureus and E. coli. More inhibitory zone was observed for the strains E. coli.
The S. aureus and E. coli that presented resistance to certain tested antibiotics, showed good susceptibility to the extracts of Triphala Masi. Variations of susceptibilities and resistance existed among same microorganisms to the same antibiotics.
Triphala Masiis widely used in the treatment of grahani (sprue), pravahika (dysentery) and raktatisara (diarrhea accompanied with blood) and is likely to be activity against the organisms associated with gastrointestinal tract. The zone of inhibition against gastrointestinal organisms such as S. aureus and E. coliconfirms that Triphala Masiis having profound antibacterial effect on these two organisms and hence effective in mitigating dysentery.
This variation observed in the activity among the market samples may be due to one or more reasons such as differences in composition of the product, differences in methods of manufacture, differences in methods of storage, differences in geographical origin and season of plant parts collected, lack of standards for formulations and lack of adequate documentation of production and testing.
From this perspective, it is of utmost importance to standardize classical Ayurvedic formulation and establish standards of testing to ensure uniformity and consistency in its activity and effectiveness. The method described may be used as the standard method for standardization of each batch of Triphala Masi, which ensures consistency and uniformity in the quality of the product.
The present study was planned to prepare Triphala Masi is used for treatment of various diseases. The prepared Masi were black colour with charcoal like taste and burnt smell. The values of analytical properties namely total ash, acid insoluble ash, water soluble ash, and loss on drying of Triphala Masi fulfill the standards criteria. pH value suggested to be acidic for Triphala Masi.
Triphala Masi exhibited a broad-spectrum antimicrobial activity against gram positive and gram negative. It inhibited the growth of S. aureus and E. coli. The above findings of pharmaceutical, analytical profile and antimicrobial study of Triphala Masi indicates that the formulation was stable and has effective antibacterial activity.
5 Conflict of interest
We declared that we have no conflict of interest.
6 Author contributions
RS and JKB have carried out the research work in the laboratory. RS compiled and analyzed the data of present work. Both authors approved the final manuscript.
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