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Trikatu Churna (TC) is an ancient traditional Ayurvedic preparation prescribed for a wide range of disorders. Though TC is an age old formulation, there are very few references on its quality control and standardization. In this work, an attempt has been made to standardize TC by qualitatively evaluating the preliminary phytochemicals. Piperine content of TC was determined using HPTLC. Evaluation of safety potential of TC samples and stability evaluation by comparative study of the in house TC formulation with marketed TC formulations with respect to their piperine content is a value addition to the current work.
Shailajan Sunita et al / IJRAP 2011, 2 (6) 1676-1678
International Journal of Research in Ayurveda & Pharmacy
ISSN 2229-3566
Research Article
Shailajan Sunita*, Sayed Neelam, Joshi Harshvardhan, Tiwari Bhavesh
F-13, Herbal Research Lab, Ramnarain Ruia College, Matunga (E), Mumabai 400 019 India
Received on: 01/10/11 Revised on: 06/11/11 Accepted on: 15/11/11
*Corresponding author
Dr. Sunita Shailajan, Associate Professor in Botany, Ramnarain Ruia College, Matunga (E), Mumbai 400 019 India
Trikatu Churna (TC) is an ancient traditional Ayurvedic preparation prescribed for a wide range of disorders. Though TC is an age old formulation, there are very few
references on its quality control and standardization. In this work, an attempt has been made to standardize TC by qualitatively evaluating the preliminary phytochemicals.
Piperine content of TC was determined using HPTLC. Evaluation of safety potential of TC samples and stability evaluation by comparative study of the in house TC
formulation with marketed TC formulations with respect to their piperine content is a value addition to the current work.
KEY WORDS: Trikatu Churna, standardization, piperine, HPTLC evaluation, safety evaluation
Trikatu Churna (TC) is an Ayurvedic polyherbal formulation of
Maricha (Piper nigrum L., fruit), Pippali (Piper longum L., fruit)
and Sunthi (Zingiber officinale Rosc, rhizome)1. Since ancient times,
it is being prescribed for Agnimandya (digestive impairment), Gala
roga (throat diseases), Svasa (asthma), Kushtha (skin diseases),
Pinasa (sinusitis), Kasa (cough) and Slipada (filariasis).
Modern bioanalytical techniques have been routinely used for
assessing the quality of the raw materials and traditional
formulations and there are reports on the standardization of some of
the herbal preparations widely used in Ayurveda2-5. Though TC is an
age old formulation, there exist meager references on its quality
control and standardization. Hence, there is a need to standardize TC
using the same.
TC (in house) was prepared in the Herbal Research Laboratory,
Mumbai using standardized raw materials as per the classical
reference1. Traditional formula composition of TC is given in Table
1. Preliminary phytochemicals of TC were evaluated as per standard
Piperine (Figure 1), a major alkaloid of Maricha and Pippali
(ingredients of TC) is reported as a bioavailability enhancer, anti-
inflammatory, anticonvulsant and antiulcer agent7-9. There are
reports on extraction of piperine using various extraction techniques
and its estimation using analytical tools from single herbs and
polyherbal formulations2,5,10,11. But, there are no methods reported
for its estimation from the complex matrix of TC. Hence, the
piperine content of TC and its ingredients was estimated using
HPTLC method was applied to evaluate the impact of storage
periods on the piperine content of stability samples and also to
comparatively evaluate marketed samples of TC with the in house
TC formulation. Acute toxicity studies were conducted to check the
safety potential of TC in mice.
Raw materials used for the preparation of TC and three different
marketed brands of TC (M-01, M-02, M-03) were procured from
Bharat Aushadhi Bhandar, Pydhonie, Mumbai and authenticated by
Herbal Research Lab, Ramnarain Ruia College. They were dried in
the incubator at 45º C for a week, powdered and sieved through 85-
mesh (BSS) sieve followed by their storage in air tight containers at
room temperature.
Standard and reagents
The organic solvents and chemicals of analytical grade were
procured from Merck Specialties Private Limited, Mumbai. Standard
piperine ( 99 % purity) was procured from Sigma Aldrich
Chemical Company, Germany.
Proximate analysis of raw materials
The quality of raw materials used in the preparation of TC was
assessed by determining the proximate parameters like foreign
matter, ash value (total ash, acid insoluble ash and water soluble ash)
and loss on drying using the standard pharmacopoeial methods12.
In house preparation of TC
TC was prepared by mixing the fine powders of Maricha, Pippali
and Sunthi in equal proportions as per the classical reference1 and
stored in air tight container at room temperature.
Preliminary phytochemical evaluation
The qualitative phytochemical tests were carried out to evaluate the
presence of major phytoconstituents in TC6.
Preparation of standard solution of piperine
Stock solution of piperine (1000.0 μg/mL) was prepared by
dissolving 10.0 mg of accurately weighed standard in small amount
of methanol and the volume was made up to 10.0 mL in standard
volumetric flask. Aliquots of 20.0-80.0 μg/mL were prepared from
this stock solution for calibration curve. Quality control samples
[LQC: MQC: HQC (25.0, 40.0 and 65.0 μg/mL)] were prepared for
precision, accuracy and ruggedness studies.
Extraction of piperine from TC, its ingredients and marketed
Extraction of marker components from TC was a daunting task
because of its complex polyherbal matrix. Hence, the extraction
conditions were optimized to achieve good fingerprinting and to
resolve piperine efficiently. Different solvents and varied solvent to
sample ratios were tested and finally TC and its ingredients (each
1.0 g) were extracted with 10.0 mL of methanol, vortexed for 1-2
min and kept standing overnight at room temperature. The mixture
was filtered through Whatmann filter paper No. 41 (E. Merck,
Mumbai, India) and the filtrate was used for further analysis. Similar
extraction procedure was followed for marketed samples of TC.
HPTLC conditions
Chromatographic separation was achieved on HPTLC plates
precoated with silica gel 60 F254 (E. Merck) of 0.2 mm thickness
with aluminum sheet support. Samples were spotted using CAMAG
Linomat IV Automatic Sample Spotter (Camag Muttenz,
Switzerland) equipped with syringe (Hamilton, 100 μL). Plates were
Shailajan Sunita et al / IJRAP 2011, 2 (6) 1676-1678
International Journal of Research in Ayurveda & Pharmacy
developed in a glass twin trough chamber (CAMAG) pre-saturated
with mobile phase. Scanning device used was CAMAG TLC
Scanner II equipped with CATS 3 software. The experimental
condition was maintained at 20 ± 2oC. Detection of piperine was
possible after derivatizing the plates with anisaldehyde sulphuric
acid reagent and photo documentation with CAMAG Reprostar 3 at
550 nm. ICH guidelines were followed for the validation of the
developed HPTLC method13.
Solvent system
Solvent system consisting of toluene-ethyl acetate-glacial acetic acid
(8: 2: 0.1, v/v/v) was used to resolve and quantify piperine from the
matrix of TC, its ingredients and marketed samples.
Estimation of piperine from TC
Samples [10.0 μL, filtrates (obtained as per section extraction of
piperine from TC, its ingredients and marketed samples)] were
applied in triplicate to a pre-coated silica gel 60 F254 HPTLC plate
(E. Merck) with the Camag Linomat IV sample spotter. The plate
was developed and analysed as per the optimized chromatographic
condition (as per section HPTLC conditions).
Method application
The developed HPTLC method was applied to study the effect of
storage on the stability of TC samples stored at different time
periods (at room temperature) in terms of piperine content. Three
different marketed samples of TC (M-01, M-02 and M-03) were
individually compared with the in-house formulation with respect to
their piperine content.
Safety evaluation
As a safety parameter, acute oral toxicity of TC was conducted on
Albino Swiss female mice weighing 18-22 g. Animals were
purchased from Haffkine Institute, Mumbai and after seven days of
acclimatization, animals were divided into two groups containing
three mice per group for the evaluation of toxicity. First group (test)
received aqueous slurry of TC orally at the dose of 2.0 g/kg body
weight of the animal while the second group (control) received 2.0
mL of distilled water. Study was conducted as per the methodology
laid down in the OECD guideline 425 viz., fixed dose procedure
(evident toxicity). Toxicity was evaluated in terms of mortality,
daily food, water intake, body weight and general behavioral
Standardization is an essential factor for ASU preparations in order
to assess their quality based on the concentration of chemical or
bioactive marker3. Modern bioanalytical techniques like HPTLC and
HPLC are being used to achieve the aforesaid objectives. In the
current work, an attempt has been made to standardize TC using
The results of proximate analysis TC and its ingredients for the
parameters like ash values (total ash, acid insoluble ash and water
soluble ash), loss on drying and foreign matter are represented in
(Table 2). It was observed that the values obtained were in
compliance with the limits documented in the Pharmacopoeia15-17. In
house preparation of TC was carried out as per the classical
reference (Table 1).
The preliminary phytochemical evaluation of TC revealed the
presence of alkaloids, tannins and essential oils, flavonoids,
glycosides and resins as major phytochemicals. Piperine content of
TC and its ingredients (Maricha and Pippali) was determined using
HPTLC technique.
The HPTLC method for estimation of piperine was validated in
terms of specificity, precision, sensitivity, recovery and ruggedness
as per ICH guidelines. Response for piperine was found to be linear
in the range of 20-80 µg/mL (r2 = 0.991) which resulted as a
regression equation y = 11.838 x -98.064. This equation was used to
determine piperine content of TC, its ingredients and marketed
samples. Developed HPTLC method was found to be precise with %
RSD < 2 % for intra-day and inter-day precision. LOD and LOQ
value for piperine was found to be 2.0 and 6.0 μg/mL respectively.
Average recovery at three different levels of piperine for formulation
was found to be 99.16 %. Method was found rugged for the
parameters like change in analyst, change in mobile phase
composition and change in spotting volume etc.
Among the various solvent systems tested, the mixture containing
toluene-ethyl acetate-glacial acetic acid (8: 2: 0.1, v/v/v) gave the
best resolution for piperine (Rf = 0.43) from the formulation matrix
which enabled its quantification as well as phytochemical fingerprint
(Figure 2). The identity of the band of piperine in TC was confirmed
by comparing its UV absorption spectra with that of the standard.
TC samples subjected to storage up to 4 months, showed variation in
piperine content. It was observed that the content of piperine
decreased on prolonged storage (Table 4, Figure 3) when compared
to freshly prepared formulation. The results of stability studies are
supported by the frequent references in classical Ayurvedic texts
regarding use of Churna18. Thus, it can be recommended that this
formulation should be consumed when fresh. The comparative study
of in-house TC formulation with other marketed formulations
revealed that the piperine content was more in the freshly prepared
in-house formulation (Table 5, Figure 4).
In acute toxicity studies, no significant change in body weight, food
intake and water intake of the animals was observed compared to
animals of control group and also no mortality was recorded. Thus,
at the dose empirically used in traditional medicine, the formulation
in the form of an aqueous slurry (2.0 g/kg body weight of animals),
can be considered with a wide margin of safety for oral use.
Such reproducible modern techniques can make the traditional
Ayurvedic medicines more acceptable in the local and global
market. Thus, rationally designed, carefully standardized, synergistic
traditional formulations and botanical drug products with robust
scientific evidence can be used as an alternative to modern medicine.
Results of the present study can be used to characterize the samples
in industry to check their uniformity. The obtained values of
physical, chemical and biological parameters for TC can be adopted
to lay down new pharmacopoeial standards to be followed in its
preparation with batch to batch consistency.
Authors are thankful to Ms. Gauri Swar for technical assistance.
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Table 1: Traditional for mula composition of TC
Sanskrit name
Botanical identity
Fruit of Piper nigrum L.
1 part
Fruit of Piper longum L.
1 part
Rhizome of Zingiber officinale Rosc.
1 part
Table 2: Proximate analysis of raw materials and TC
Foreign matter
Total ash
Water soluble ash
Loss on drying
% Mean (n = 3) ± S. D.
0.463 ± 0.036
4.276 ± 0.080
2.360 ± 0.051
14.856 ± 0.427
1.557 ± 0.030
3.987 ± 0.060
2.093 ± 0.034
12.076 ± 0.308
0.350 ± 0.037
5.176 ± 0.036
3.433 ± 0.043
9.596 ± 0.355
Not applicable
5.765 ± 0.200
3.216 ± 0.037
9.273 ± 0.012
Table 3: Piperine content in TC and its ingredients using HPTLC
Piperine content (mg/g)
[Mean ± S. D., n=3]
3.53 ± 0.12
1.65 ± 0.24
3.14 ± 0.55
Table 4: Stability study of TC in terms of piperine content using HPTLC
Storage period
Piperine content (mg/g)
[Mean ± S. D., n=3]
3.14 ± 0.55
2.48 ± 0.06
0.50 ± 0.10
Table 5: Comparative study of in-house TC and marketed formulations in terms
of piperine content using HP TLC
Name of formulation
Piperine content (mg/g)
[Mean ± S. D., n=3]
In-house TC
3.14 ± 0.55
2.24 ± 0.11
2.67 ± 0.04
2.88 ± 0.17
Figure 1: Structure of piperine
Figure 2: HPTLC detection of piperine from TC and its ingredients at 550 nm, Track
details - 1: Maricha, 2: Pippali, 3: Piperine, 4: Sunthi, 5: TC
Figure 3: Stability samples of TC on HPTLC at 550 nm, Track details - 1: 0 month
sample, 2: 2 month sample, 3: piperine, 4: 4 month sample
Figure 4: In house and marketed samples of TC on HPTLC at 550 nm, Track details -
1: TC, 2: M-01, 3: piperine, 4: M-02, 5: M-03
Source of support: Nil, Conflict of interest: None Declared
... The solvent system consisting of tolueneethyl acetate-glacial acetic acid (8:2:0.1, v/v/v) was used to resolve piperine from the matrix of TC (45)(46)(47). Densitometric determinations were performed in fluorescence mode by CAMAG TLC scanner. ...
... If maintained under the same conditions, HPTLC analysis shows that all the three batches of TC have an Rf value of 0.43, indicating the presence of Piperine (45)(46)(47). Piperine is . ...
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Trikatu Churna (TC) comprising Zingiber officinale rhizome, Piper longum, and Piper nigrum fruit, is effective in treating liver diseases and has high nutraceutical values. However, the efficacy of TC in treating alcoholic liver disease (ALD) and its mechanism remain largely unknown. This study evaluated the hepatoprotective effects of different doses of TC as well as to identify the bioactive components and determine their mechanism of action against ethanol-induced ALD. A compound-target network analysis model of TC was established to identify its potential bioactive compounds and pathways that might regulate its hepatoprotective effects. Further, in-vivo studies were performed to validate the potential of TC (200 mg/kg and 400 mg/kg b.w.) in the treatment and management of ALD. The study revealed that both the dosages of TC demonstrate significant (p > 0.0001) hepatoprotective effects by improving body weight, total bilirubin, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphate (ALP), total cholesterol, total protein, globulin, albumin, and liver morphology. The High-performance thin-layer chromatography (HPTLC) fingerprinting of TC showed the presence of piperine. Network pharmacology identifies the role of TC in regulating various signaling processes including Advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE), Hypoxia-inducible factors (HIF-1), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Kappa B), and Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling to exert its anti-inflammatory, antioxidant and anti-apoptotic role in managing ALD. Based on the bioinformatics analysis, some of the key targets of TC were found to be Prostaglandin-Endoperoxide Synthase 2 (PTGS2) or Cyclooxygenase-2 (COX-2), Sirtuin 1 (SRT1), and caspase-3. These effects may serve as a novel therapeutic option for the treatment of ALD. These preclinical validation studies for the ethnopharmacological potential of TC in ALD treatment further paved the way for researchers to perform next-level translational and clinical studies. Further, in-depth experimental studies for the validation of these bioinformatics-based results will give a clearer picture of mechanisms.
... Dahikar et al. [3], Malvankar and Abhyankar [4] studied the in vitro antibacterial potential of Trikatu churna and its ingredients. Sunita et al. [5] studied the standardization of Trikatu churna using bioanalytical tools. The preliminary phytochemical screening showed the presence of alkaloids, tannins and essential oils, flavonoids, glycosides, and resins. ...
Full-text available
“Trikatu” is an Ayurvedic proprietary medicine. It is the mixture of fruits of Piper nigrum, Piper longum and rhizome of Zingiber officinale. This combination is found to be active against a wide range of health problems. The present study is to evaluate the phytochemical characterization and antibacterial activities of “Trikatu”. The fruits of P. nigrum, P. longum and rhizome of Z. officinale were collected from local areas of Wayanad and Ernakulam districts of Kerala, India. Collected plant materials were washed and shade dried. The dried plant materials were then ground to fine powder and stored. Distilled water, methanol, ethanol, acetone and diethyl ether were used for extraction of the powdered samples. Analysis of phytochemicals were done using standard methods. The extracts showed the presence of flavonoids, alkaloids, phenols, tannins, quinine, cardiac glycosides, saponins, steroids, carbohydrates etc. The best results are obtained in methanol and ethanol extracts while diethyl ether extracts showed minimal presence of the compounds. Antibacterial activity was evaluated using agar well diffusion method against four strains of bacteria. Methanol and diethyl ether extracts showed maximum inhibitory activity against all test organisms.
... e the siddha Formulary of india, f Boericke (1993), g Gautam et al. (2020). (Shailajan et al. 2011). It is mentioned in ayurvedic literature and is widely used by ayurvedic practitioners for its primary effect on the upper GI tract . ...
Translation of traditional knowledge of herbs into a viable product for clinical use is still an uphill task. Piperine, a pungent alkaloid molecule derived from Piper nigrum and Piper longum possesses diverse pharmacological effects. Traditionally, pepper is used for arthritis, bronchitis, gastritis, diarrhea, snake bite, menstrual pain, fever, and bacterial infections, etc. The anti-inflammatory, antioxidant and immunomodulatory actions of piperine are the possible mechanisms behind its therapeutic potential. Various in-silico and experimental studies have shown piperine as a possible promising molecule in coronavirus disease (COVID-19), ebola, and dengue due to its immunomodulatory and antiviral activities. The other important clinical applications of piperine are due to its bio enhancing effect on drugs, by modulating, absorption in the gastrointestinal tract, altering activities of transporters like p-glycoprotein substrates, and modulating drug metabolism by altering the expression of cytochrome P450 or UDP-glucuronosyltransferase enzymes. Piperine attracted clinicians in treating patients with arthritis, metabolic syndrome, diabetes, skin infections, gastric and liver disorders. This review focused on systematic, evidence-based insight into the use of piperine in clinical settings and mechanistic details behind its therapeutic actions. Also, highlights a number of clinical trials of piperine at various stages exploring its clinical application in cancer, neurological, respiratory, and viral disease, etc.
... The Ayurveda Formulary of India has also given the period as the time from the date of manufacture within which the formulations should be consumed for best results. [5] By following prevalent guidelines, the stability study of some Ayurveda formulations such as powder, [6,7] pills, [8] supplements, and confectionaries [9] has been attempted earlier. ...
... Trikatuchurna: Trikatu Churna is well known Ayurveda Formulation, comprised of the fruits of two medicinal important plants of Piper longum (Pipali) along with Piper nigrum (Marica) and rhizomes of Zingiber officinalis (Saunth) (Shailajan et al., 2011). ...
... [19] Sunita et al., 2011, have used various bioanalytical tools to standardize Trikatu Churnam. [20] ...
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Introduction: The present study deals with the gas chromatography-mass spectrometry (GC-MS) analysis of one Ayurvedic digestive powder, Trikatu Churnam. Materials and Methods: The Trikatu Churnam was procured for standard Ayurvedic vendor at Chennai and was subjected to GC-MS analysis by the standard as per the standard procedure. Results: The GC-MS profile indicated the presence of some important biomolecules such as longipinocarveol, trans-., beta-bisabolene, caryophyllene oxide., alpha.-ylangene, dodecanedioic acid, ursodeoxycholic acid, 1(2H)-naphthalenone, octahydro-4a,8a-dimethyl-7-(1-methylethyl)-, [4aR-(4a.alpha.,7.beta.,8a.alpha.)]-, 3-methyl-1-adamantaneacetic acid, thiazole-2,4(3H,5H)-dione, 5-benzylideno-3-[(ethylphenylamino)methyl]-, and i-propyl 5,9,17-hexacosatrienoate, which has medicinal roles indicating the efficacy of Trikatu Churnam as a medicine for digestive disorders. Conclusions: The above results and discussion indicates that Trikatu churnam is a good formulation for digestive disorders.
... [97] However, there are PIPs marketed in the form of ayurvedic formulation: Habb-e-Khardel. [98] Eladi Gutika, [99] Jawarishe-Bisbasa, [100] Trikatu Churna, [101] LasunadiVati, Marichyadivati and Kaphaketu rasa. [102] Drug delivery systems One of the limitations of PIP is its low solubility in water, causing limitation in clinical application. ...
Piperine (PIP) is a natural alkaloid isolated from Piper longum L. that presents antioxidant, anticonvulsant, antimicrobial, neuroprotective, larvicidal, antiparasitic, anticancer effect and other pharmacological properties. However, the low aqueous solubility is the main barrier to its development from the laboratory to the clinic as a drug. Several strategies have been used to overcome this obstacle, like the incorporation of PIP into different drug delivery systems turned out to be highly efficient. In addition, several methods for the quantitative and qualitative analysis of PIP in various raw materials, including biological fluids (plasma, urine, metabolites, brain), plants and drug delivery systems, were investigated. Most recently high-performance liquid chromatography was used together with several detectors for this purpose. Therefore, this review presents a summary of characteristics chemical and biological properties of PIP as well as several techniques and analytical methods to optimize the analytical signal, increase sensitivity, selectivity and reduce the effects of interference for this drug.
The Chemistry inside Spices & Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included. Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin. Audience: This book is an ideal resource for scholars (in life sciences, phytomedicine and natural product chemistry) and general readers who want to understand the importance of herbs, spices and traditional medicine in pharmaceutical and clinical research.
From ancient times, phytopharmaceuticals have played an important role in the management of human health. Piperine, an alkaloid with the piperidine nucleus was discovered and isolated by Hans Christian Ørsted, from the fruits of Piper nigrum. Piperine forms is slightly water soluble and forms monoclinic needles and possess a strong pungent taste. Piperine contains plentiful established health effects and beneficial therapeutic properties. Cells and enzymes are key elements in biotechnological processes to carry out a wide variety of very specific reactions under judicious conditions to produce piperine and their products. Piperine also serves as bio-enhancers in conjunction with drugs to stimulate drug molecules’ activity across different routes by improving the drug's bioavailability across the membrane, raising the drug's effect across conformational interaction, and working as a drug receptor. In recent years, there has been significant interest in the use of piperine to treat many illnesses, its health-beneficial effects, and its work as bio-enhancers. Due to their biological activity, piperine has the potential to be used in health and medicine.
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Background: Shelf life is the length of time, after which all substances starts to degrade in their qualities and become unsuitable for consumption or sale. Every product has definite shelf life, which depends on various physical, chemical, environmental, and biological factors. Shelf life of various compound formulations was clarified in the classical text. So there is a need to revalidate and establish the shelf life of individual formulation. As the Government of India made it mandatory to display in the labels the shelf life of all products, it becomes essential to evaluate the actual shelf life of individual products by following the specified guidelines. Aim: To evaluate the shelf life of Shirishavaleha (SHA) and its granules prepared with Khanda Sharkara through accelerated stability parameters. Materials and methods: Physicochemical parameters of SHA and Shirishavaleha granules (SHG) were calculated by maintaining 40°C ± 2°C temperature and 75 ± 5% relative humidity at the intervals of 0, 1, 3, and 6 months. Based upon the observations, intercept, slope, 10% degradation of the sample, and finally the shelf life were calculated. Results: Accelerated stability study reveals the shelf life of SHA as 5.9 years, while that of the granules as 5.3 years. Similar R f values obtained in high-performance thin-layer chromatography (HPTLC) analysis of both samples initially and after 6 months showed minimum deterioration of the product. Microbial count and heavy metals were below the permissible limits in both the samples. Conclusion: Guidelines specified in the Drugs and Cosmetics Act, 1940, mentions 3 years as the shelf life of Avalehas and granules. This time period is general, and different formulations may have different shelf lives based upon the factors such as their composition, etc. The samples tested in the current study, i.e., SHA and SHG, are found to have a shelf life of 5.9 and 5.3 years, respectively, through accelerated stability study that are almost double the average time specified in the guidelines.
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Vasavaleha is a traditional Ayurvedic oral Herbal formulation consisting of five herbs, Vasaka (Adhatoda vasica Nees.), Pippali (Piper longum Linn.), Sugar, Ghee and Honey. It is available as a popular proprietary, from most manufacturers of ayurvedic drugs. A selective, precise and accurate High Performance Thin Layer Chromatography (HPTLC) method has been developed for the simultaneous quantification of Vasicine and Piperine in Vasavaleha as well as its bulk drug. The method employed TLC aluminum plate precoated with silica gel 60 F254 as a stationary phase. The solvent system consists of Dioxane: Toluene: Ethyl acetate: Methanol: Ammonia (1.5:2:1:1:0.3% v/v). This system was found to give compact spot for Vasicine and Piperine. Densiometric analysis was carried out in the absorbance mode at 285 nm. The linear regression analysis data for the calibration plot showed good linear relation with r2 = 0.992 and 0.993 with respect to peak area for Vasicine and Piperine respectively, in concentration range 2-10 μg/spot. The method was validated for precision, recovery, Limit of Detection and Limit of Quantification. The proposed HPTLC method was found to be simple, precised and accurate and can be used for the quality control of the raw materials as well as formulations.
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A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of Piperine in capsule dosage forms. The stationary phase used was precoated silica gel 60F 254. The mobile phase containing Toluene and Methanol in proportion of 80:10 v/v was used to separate the spot of Piperine. The detection of spot was carried out 332nm. The Rf value was found to be 0.49± 0.01. The method was validated in terms of Linearity, Accuracy and Precision. The linearity curve found to be linear in between 10-45 ng/spot. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 1.23ng and 9ng. The proposed method can be used to determine the drug content from marketed formulations.
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The concept of Saviryta Avadhi (shelf life) for different Ayurvedic dosage forms are not specified in major classics like Charaka, Sushruta, etc. but after 13(th) century, it was considered in various authentic Ayurvedic texts like Vanga Sen, Shrangdhar Samhita and Yogaratnakar. Along with the indication of best before use duration, the importance of quality of package and storage condition is also emphasized in classics. Nowadays, due to development and adaptation of packaging and storage technology by Ayurvedic industries, a need arise to re-study and re-establish the newer ones criteria. Recently, Government of India has also issued a Gazette notification and proposed the shelf life of the Ayurvedic formulations, which seems to be unscientific and the authority has not properly considered the current development in this field. There is also a need to develop a specific guideline for the assessment of shelf life of Ayurvedic drugs. Some of the studies show that the shelf life of these types of dosage forms,can be increased by utilizing current advanced pharmaceutical technologies. Through the paper, a trial is made to explore the knowledge of shelf life of Ayurvedic drugs in classics, the current efforts and the potential need.
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Hingvadi Taila (HT) is an Ayurvedic formulation which is used for Karnashoola (ear-ache) since ancient times. In the present study, the quality of raw materials, intermediates obtained during the preparation of Hingvadi Taila and the final product was assessed by modern scientific procedures. Values of proximate analysis of the raw materials used were found to be within the permissible pharmacopoeial limits. Hingvadi Taila was prepared using Shodhita Hingu, Shunthi, Tumburu and Jala in Sarsapa Taila as base oil as per Ayurvedic Formulary of India (AFI). Preliminary phytochemical evaluation of Hingvadi Taila revealed the presence of essential oils, tannins and glycosides while flavonoids, alkaloids and resins were found to be absent. Physicochemical parameters like acid value, saponification value and peroxide value for both Hingvadi Taila as well as Sarsapa Taila have been evaluated. A simple, rapid, accurate and sensitive Reverse Phase-High Performance Liquid Chromatographic method (RP-HPLC) was developed and validated for the quantitation of ferulic acid from Hingu, Shodhit Hingu and Hingvadi Taila. Skin irritation test of Hingvadi Taila was carried out on Rabbits (New Zealand Strain) to check its safety. No erythema, oedema and irritation were observed up to observation period of 72 hours.
A simple, rapid and precise reverse-phase high performance liquid chromatographic method has been developed for the quantitative determination of piperine from Ayurvedic Polyherbal formulations Arkavati, Krvyadras and Marichyadi taila. Chromatographic analysis was carried out on cosmosil C18 column (150mm x 4.6mm, 5μm particle) with a mobile phase of methanol: water in the volume ratio of 70:30 at a flow rate of 1.0 mL min-1. Quantitation was performed using a PDA-detector at 342 nm. Linear response for piperine was obtained over a range of 200 to 3000 ng mL-1. The method was validated for linearity, precision, accuracy and can be effectively used to evaluate quality of Arkavati, Krvyadras and Marichyadi taila.
Caesalpinia bonduc (Linn.). is commonly called as latakaranj and is widely distributed throughout India. A sensitive, simple, and accurate High-Performance Thin-Layer Chromatographic method has been established for quantitation of β-sitosterol from seed powder of Caesalpinia bonduc (Linn.) collected from different regions of India as well as from Menstrowin tablets, a polyherbal formulation used against female reproductive disorder. The seed powder was extracted with Methanol and used for quantitation. The concentration of β-sitosterol was found to be 0.1134 mg/gm. in the seed powder of Caesalpinia bonduc (Linn.) collected from Mumbai which was used as a reference sample. Quantitation of β-sitosterol was also carried out for Caesalpinia bonduc (Linn.) seeds collected from different regions of India like Kutch (Gujrat), Malvan, Madhya Pradesh and Raigad and the concentrations were found to be 0.2080 mg/gm, 0.1432 mg/gm, 0.1244 mg/gm and 0.0820 mg/gm respectively. The concentration of β-sitosterol from polyherbal formulation Menstrowin (Safe Life Herbals Pvt, ltd.) was found to be 0.02 mg/gm. Quantitation was carried out on HPTLC silica gel 60 F 254 pre-coated plates with the mobile phase Toluene: Ethyl Acetate: Methanol (7:1:0.5) (v/v/v). A TLC scanner set at 366nm in fluorescence/reflectance mode was used for quantitation. β-sitosterol response was linear over the range 5 μg mL -1 to 50 μg mL -1. The method was validated for linearity, precision, accuracy and robustness.
Piperine, a characterizing compound present in fruits of Piper nigrum and Piper longum used as bioavailability enhancer. Ingredients of antioxidant, anti-inflammatory activity has been extracted using soxhlet and supercritical fluid extraction technique. It was isolated using column chromatography. Characterization of compound was done by spectroscopic technique. A simple, rapid, accurate and specific HPTLC method developed and validated. The method proposed can be used for the routine analysis of both P.nigrum and P.longum fruit material and its formulations.
Piperine, a major alkaloid of Piper longum and Piper nigrum has been reported to have several pharmacological/toxicological effects. Though a number of methods for analysis of this omnipresent food component in pepper fruits are available, its analysis in body fluids has been largely neglected. A high-performance liquid chromatography method for the analysis of piperine in rat plasma is presented in this communication. Analysis was performed using a Symmetry C(18) column (250x4.6 mm) by isocratic elution with 25 mM KH(2)PO(4) (pH 4.5)-acetonitrile (35:65) and UV detection at 340 nm. The calibration plot was linear over the range studied (2-2000 ng) with correlation coefficient of 0.9984. Limit of detection and limit of quantitation were 1 ng/ml and 3 ng/ml, respectively. Good overall recovery (85.5+/-6%) was obtained with 4 ml ethyl acetate and extraction time of 3 min. Intra- and inter-assay coefficient of variation was found to be less than 7.5%. Plasma concentration-time profile of piperine in a conscious rat implanted with jugular vein cannula was obtained using this method. The method is simple, sensitive and reproducible.