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HPLC method validation for simultaneous estimation ofmadecassoside, asiaticoside and asiatic acid in Centella asiatica
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... One reason is the absence of validation of the process used. Current interest is no longer based solely on the isolation of active compounds and investigation of their bioactivities, but also on the validation of analysis methods that play an important role in controlling or guaranteeing the quality of a product (Tiwari et al., 2010). ...
... In validating the analytical method, the method used must be simple, inexpensive, the time required is short, precise, accurate and can be used widely (Tiwari et al., 2010). One such method is Ultra-Performance Liquid Chromatography (UPLC). ...
Andrographis paniculata is an herbaceous plant belonging to the family Acanthaceae dan also known as the “King of Bitters”. It has been utilized as traditional medicines by the local people in India and Southeast Asia. Many studies have been reported regarding the pharmacological activities of A. paniculata and Andrographolide. Currently, in addition to compound isolation and bioactivity assays, validation of analysis method is also required. Validation is used to demonstrate that the method used is appropriate and the results are precise and accurate. The method used needs to be simple, concise, precise, accurate, and widely used. The analysis in this study was carried out using Ultra-Performance Liquid Chromatography because the instrument operates at high pressure. In addition, the system uses fine particles which can reduce column length as well as solvent and time consumption. This study aims to validate analytical methods in determining the contents of andrographolide in an A. paniculata Leaves. The analytical method was validated through a Waters Alliance UPLC System with photodiode array (PDA) detector. The A. paniculata extract and standard solutions of Andrographolide were analyzed using reverse-phase C18 column which was maintained at 40°C. A mobile phase of 0.1% formic acid in acetnotirile and 0.1% formic acid in water was used at flow rate of 0.3 mL/min to achieve gradient elution. The linearity of the andrographolide showed excellent results (R2= 0.9999) in the concentration range of 7.8–250.0 µg/mL. The LOD and LOQ values of andrographolide were 0.068 µg/mL and 0.205 µg/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) and relative error (RE) accuracy values of andrographolide were <±15. The quantitative analysis found that A. paniculata extract contained 12.45 ±0.06 mg/g of Andrographolide. This developed UPLC-PDA method was proven to be precise, specific, sensitive, and accurate for routine quality assessment of raw material of Andrographis paniculata leaves content.
... High-performance liquid chromatography (HPLC) technique has emerged as the primary methodology for AC analysis. Several methods for determining AC utilizing reversed phase HPLC in either gradient [13] or isocratic mode [14][15][16][17] have been published in the literature. All the reported methods are expensive and time-consuming. ...
The purpose of this study was to develop a rapid, sensitive, and precise RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) method for the estimation of asiaticoside (AC) using the analytical quality by design (AQbD) approach. Initially, important AQbD prerequisites like analytical target profile (ATP) and critical analytical attributes (CAAs), such as theoretical plates and tailing factor, were defined. The final chromatographic conditions for the analysis of AC consist of stationary phase {A COSMOSIL 5C18-MS-II packed column (250 mm × 4.6 mm i.d, 5 µm)}, mobile phase was Smix {Methanol and Acetonitrile (40:25)}, and Orthophosphoric acid buffer (0.025 % OPA) in the ratio of 65:35 v/v at a flow rate of 1.0 mL/min and detection wavelength was 205 nm. The diversity in CAAs with different inputs was explained using the Ishikawa fishbone diagram. Taguchi design was selected as the first screening design to choose the critical material attributes (CMAs) that influence the method development. Subsequently, for more systematic optimization of the chromatographic technique and evaluation of CAAs, central composite design (CCD) was employed. In conclusion, the findings of the present study validated the utility of AQbD in the systemic design of a liquid chromatographic method with fine sensitivity for AC estimation in pharmaceutical products.
... The total run time was set at 16 min. The %label amount of AA in the tablets was estimated from the regression equation of the AA calibration curve [40,41]. ...
The objective of this study was to develop effervescent cleansing tablets that can be dissolved and turned into liquid soap, which can be used for bathing or soaking the body. The asiatic-acid-loaded solid lipid microparticles (AASLMs) were prepared via the hot emulsification method followed by cold re-solidification and then freeze-dried to obtained dry powder. The physicochemical properties such as morphology and % entrapment efficiency (%EE) were evaluated. The results revealed that AASLMs have an irregular shape, and the %EE for the resulting AASLMs was 92.04 ± 3.43%. The tablets were manufactured via the direct compression technique. The compatibility test was conducted to ensure that the excipients are compatible with the active ingredient. The angle of repose, Carr’s index, and Hausner’s ratio were studied to evaluate the flowability of the powder blend before compression. The weight of each tablet was set to 1000 mg, and physicochemical characteristics, in vitro dissolution, ex vivo cleansing efficacy, and stability were evaluated. The results showed that the active ingredient was compatible with other excipients, as the results obtained from FTIR spectra indicated the absence of potential chemical interaction between the active ingredient and excipients used in this study. Additionally, all formulations had good flow properties. The effervescence times of selected formulations, F2 and F3, were <5 min, with favorable pH and hardness values. The friability values of all formulations exceeded 1% because the excipients used in effervescent tablets are very fragile. The release of asiatic acid (AA) from the tablets was dependent on the concentration of SLS. In an ex vivo test, it was discovered that the developed products F2 and F3 showed much more effective cleansing efficacy than water. Nevertheless, brown spots appeared in the tablets and the AA content was significantly decreased in both tested formulations after 3 months’ storage at 40 ± 2 °C/75% RH ± 5% RH. The stability study revealed that the developed products were not stable at high temperature and humidity. Therefore, it is recommended that the developed effervescent tablets are not stored at a high temperature.
... Various advanced analytical techniques like HPTLC, HPLC, and LCMS have been effectively employed to qualitatively and quantitatively Surabhi Tiwari et al. analyse the bioactive phytocompounds in C. asiatica. (Jain and Agrawal, 2008;Tiwari et al., 2010;Sharifuldin et al., 2013;Kabir, 2014;Srivastava et al., 2015;Monton et al., 2018;Srivastava et al., 2019;Rafamantanana et al., 2019;Nair et al., 2012;Zakaria et al., 2019). These constituents play a dynamic role in the authentication, identification, and standardization of bio-markers, adulterants, and quality checks, of raw materials and products in various pharma industries. ...
... In the context of drug manufacturing and laboratory testing, validation is essential to comply with current good manufacturing practices (CGMP) and good laboratory practices (GLP) requirements (Chikanbanjar et al., 2020). A more practical method for determining plant constituents is necessary, which is less complicated, affordable, time-efficient, and can be used on a larger scale (Tiwari et al., 2010). Ultra-Performance Liquid Chromatography (UPLC) is a potential method that can be utilized for the estimation of plant constituents. ...
Cassia angustifolia is a commonly found wild plant from the family Caesalpinaceae that originates from Yemen and Hadramaut province in Southern Arabia, where it is called Arabian senna. The leaves of the plant have been used to treat a variety of ailments such as constipation, malaria, anemia, loss of appetite, and indigestion. Sennosides A and B are the major glycosides found in the leaves and pods of C. angustifolia and are important ingredients in purgative medicines. These compounds are considered as the major active components of Cassia plants and are responsible for their therapeutic activities. To assess the quality and quantity of sennosides A and B, an appropriate analytical method is required, which must be simple, accurate, precise, and widely used. The UPLC-ESI-MRM/MS method was used in this study to validate the analytical method in determining the contents of Sennoside B in senna leaves extract. The validation parameters included specificity, system suitability, linearity, sensitivity (LOD, LOQ), accuracy, and precision. The results indicated that the optimization of MRM using the direct infusion method provided good separation when eluted using liquid chromatography. The validation parameters for system suitability obtained RSD under 2%. The linearity of sennoside B showed excellent results (R2 = 0.999) in the concentration range of 0.98–62.5 µg/ml. The LOD and LOQ values of sennoside B were 0.011 µg/mL and 0.034 µg/ml, respectively. The accuracy values of sennoside B met the predetermined criteria, with RSD < 2% and % recovery of 97-102%. The quantitative analysis revealed that Cassia angustifolia extract contained 0.43 ±0.06 mg/g of sennoside B.
... Among all the hematological cancers, leukemias are among the most typical types, where leukemic blood cells increase in large numbers (Azim et al., 2010;Schlenk et al., 2008). According to recent study, asiatic acid extracted from C. asiatica is employed as a potential treatment against leukemic malignancies (Charles Dorni et al., 2017;Lucas et al., 2010;Tiwari et al., 2010;Wu et al., 2015). The cytotoxic activity of asiatic acid against HL-60 (IC 50 46.67 ...
Centella asiatica, a pre-historically utilized medicinal herb that is well recognized as a traditional phytotherapeutic substance for treating a variety of diseases. However, its phytochemicals use as a functional component in food and beverage has recently attracted the attentions of food researchers and scientists. C. asiatica possesses pharmacological value due to the presence of phytochemicals like flavonoids and terpenoids, mainly asiatic acid, asiaticoside, and madecassoside. Its role as a functional component in developing various processed food products and packaging films is continuously being explored. It improves the micronutrient profile and antioxidant potential of developed food products like noodles, chocolates, soups, pasta, etc. However, comprehensive scientific validation based on clinical trials is inadequate, which impede the perception of health benefits and nutritional importance of C. asiatica rich food products. Furthermore, there is a need to extensively examine the composition of C. asiatica active compounds in order to maximise its application in the food processing and preservation industry. Therefore, the goal of this paper is to investigate into the existing information on the extraction, identification, and quantification of C. asiatica phytochemicals, as well as to explore their biological activity. The potential of C. asiatica for developing functional foods and beverages, as well as their processing and preservation to improve human health, is also highlighted.
... The UV detection wavelength was set at 210 nm. The compounds were identified by comparing their retention times and UV spectra with those of the markers (Tiwari et al., 2010). ...
Centella asiatica (Linn) Urban, is an important medicinal plants and commonly known as Brahmi or Mandookaparni belongs to the family Apiaceae. The present investigation was undertaken with a major objective of developing autotetraploid genotypes of Brahmi, employing the techniques of ploidy breeding, especially induction of autotetraploidy using colchicine, which is quick, reliable and very effective in high yield of saponins. Centella asiatica was used as experimental plant material and they treated of various concentration colchicine solution (0.1% to 1.0%) with different time durations (1to 8hrs.). I observed that, 0.2% colchicine concentrations with 4 hour durations were effective concentrations for inductions of autotetraploidy as compared to other. It confirm through morphological and anatomical characteristics of autotetrapoids and diploid plants. Then, C. asiatica estimation of spaonins of autotetraploidy was analyzed through HPLC method. It found that, the induced autotetraploids (colchiploids) showed 1.30 times high levels of medecassic acid, terminolic acid and asiatic acid (saponins) as compared to normal diploid plants. Thus the present investigation, establishes that it is possible to improve the quantity of therapeutically active compounds through polyploidy breeding by using colchicine treatment of medicinal plants.
... Pada hewan percobaan, kemampuan menstimulasi susunan syaraf pusat berhubungan dengan bertambahnya aktivitas lokomotor (Nikajoo, 2009). Aktivitas lokomotor merupakan aktivitas gerak yang dapat menstimulasi syaraf pada otak (Tiwari, et al. 2010). Tonik dapat digunakan untuk menstimulasi sistem syaraf pusat (Mutschler, E., 1986). ...
Pegagan (Centella asiatica (L). Urb) dikenal secara empiris sebagai obat tradisional untuk mempercepat aktivitas syaraf, meningkatkan daya ingat,dan tonik untuk organ tubuh(hati, ginjal, otak). Efek tonik dapat ditentukan dengan menggunakan metode natatoryexhaustion melalui pengamatan efek stimulansia suatu obat pada hewan uji. Tujuanpenelitian ini adalah untuk mengetahui efek tonik ekstrak etanol herba pegagan pada mencitjantan (Balb/C) dan menentukan dosis efektif yang menunjukkan kemampuan mencit untukmempertahankan diri ketika direnangkan. Penelitian ini menggunakan lima kelompok uji,tiap kelompok terdiri atas lima mencit jantan. Kelompok kontrol positif, kontrol negatif,kelompok dosis I, II dan III ekstrak etanol herba pegagan diberikan masing-masing kafein100 mg/kgBB, CMC 0,5%, 50 mg/kg BB, 100 mg/kg BB, dan 150 mg/kg BB secara peroral.Pengujian efek tonik dengan metode natatory exhaustion ditunjukkan dengan bertambahnyawaktu kemampuan mencit untuk mempertahankan diri ketika direnangkan. Pertambahanwaktu tersebut menunjukkan peningkatan daya tahan mencit. Dosis terbaik yang dapatdigunakan sebagai tonik adalah 100 mg/kg BB.
... There have been several analytical methods developed using high-performance liquid chromatography (HPLC) with ultraviolet detection at 205 or 210 nm reported for the determination of the four triterpenoids (asiaticoside, madecassoside, asiatic acid, and madecassic acid) from C. asiatica (L.). [32][33][34][35][36] Most of these analytical methods are based on reverse-phase chromatography with C18 silica gel as stationary phase and invariably having a chromatographic run time of 30-85 min. Chromatographic resolution has been the area of concern for quantifying the two triterpenoid glycosides and their aglycones found in C. asiatica (L.). ...
The aim of the study is to standardize a simple and an effective extraction method for industrial preparation of Centella asiatica (L.) extract rich in triterpenoids. Macroporous resin purification process was adapted to enrich the triterpenes extracted from the herb. A sensitive analytical method with good resolution, linearity and a shorter run time of 10 minutes was developed in UHPLC-Q-ToF-MS for monitoring the triterpenes (madecassoside, asiaticoside, madecassic acid and asiatic acid) content at each stage of the extraction process. The standardized process could enrich the triterpene, madecassoside in the product up to a purity of 72.51% with overall recovery of the compound to 90.79%. A purified form of asiaticoside with a purity of 85% was obtained by dealcoholization and precipitation process exploiting the differential water solubility of the two major triterpenes. The binding and recovery of the aglycones (madecassic acid and asiatic acid) were observed to be poor and hence the overall recovery of aglycones by this process was found to be very low. The purified triterpenoids were also confirmed by the ¹H NMR. Thus, the results suggest that the standardized process can be converted to industrial scale preparation of high value C.asiatica (L.) products rich in madecassoside or asiaticoside.
Technical report on ethnomedicinal use of P. foetida. Paederia foetida Linn. (Rubiaceae), known as Prasarini‘ in Sanskrit is an extensive foetid smell climber. It has been used for various purposes in the Indian systems of medicine as well as in folk-lore medicine (Table 2). The tribal community of Northeast part of India and other parts of Asia including Bangladesh use this plant mainly for gastrointestinal diseases. Our aim is to find out a most active standardized fractional extract which could be formulated for the treatment of peptic ulcer disease (PUD). Chemical modification of the most active fraction will also be done with an aim to increase the potency/efficacy of the extract.
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