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Adensitometric HPTLC method has been established for analysis in alcohol extracts of the seeds and leaves of Achyranthes aspera L. The extracts were separated on aluminum foil-backed silica gel 60 F 254 plates. Detection was by measurement of absorbance at 490 nm, in absorbance/reflectance mode, after derivatization with 10% H 2 SO 4 in 95.5% ( v/v ) ethanol. The R F of oleanolic acid was 0.22. The oleanolic acid content of both extracts was compared statistically and the amount in the seed extract found to be higher (0.970%) than that in the leaf extract (0.666%).

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A sensitive and reliable high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantitative determination of oleanolic acid in the dried roots of Helicteres isora Linn. Total sapogenins were isolated from the roots, and their alcoholic extract was applied on silica gel G 60 F254 plates with toluene-ethyl acetate-glacial acetic acid, 7:3:0.1 (ν/ν/ν) as mobile phase. Detection and quantification were performed by densitometric scanning at 529 nm. The accuracy of the method was confirmed by conducting recovery studies at three different levels using the standard addition method. The average recovery of oleanolic acid was found to be 98.98%. The proposed HPTLC method provides good resolution of oleanolic acid from other constituents present in sapogenin extract of dried roots of H. isora and can be used for quantification of oleanolic acid present in the extract. The method is simple, accurate, and precise.

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A sensitive, simple, and accurate high-performance thin-layer chromatographic method has been established for determination of oleanolic acid in whole-plant powder from Oldenlandia corymbosa L. A methanol extract of the powder was used for the experimental work. The concentration of oleanolic acid in whole plant was found to be 1.892 μg g −1 . Separation was performed on aluminum HPTLC plates coated with silica gel 60 F 254 , with dichloromethane-toluene-acetone-methanol, 3 + 4 + 1.5 + 0.3 ( v/v ), as mobile phase. After development, plates were treated with Liebermann-Burchard reagent and detection and quantification were performed by densitometry at λ = 529 nm. Detection and quantitation limits were 0.1 μg and 0.5 μg, respectively. Oleanolic acid response was linear over the range 1 to 9 μg. The validated HPTLC method can be used for routine quality-control analysis of Oldenlandia corymbosa L. whole-plant powder and for quantitative determination of oleanolic acid.

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Oldenlandia corymbosa Linn. (Rubiaceae) is an important herb traditionally used as a febrifuge and liver tonic. In this study, a high-performance thin-layer chromatography (HPTLC) method has been established for the quantification of four bioactive markers, oleanolic acid (OA), ursolic acid (UA), lupeol (LU), and stigmasterol (ST), in the whole plant of O. corymbosa. Separation was achieved on silica gel 60 F254 HPTLC plates using hexane-ethyl acetate-methanol (8.2:1.8:0.5, v/v) for oleanolic acid and ursolic acid; and toluene-methanol (9.4:0.6, v/v) for lupeol and stigmasterol as the mobile phases. The quantitation of the four markers was carried out using the densitometric scanning at 540 nm after derivatization using sulfuric acid reagent. The linear regression analysis data for the calibration plots showed a good linear relationship (r 2 = 0.9831–0.9979) in the concentration range of 1200–4200 ng for oleanolic acid, 400–1400 ng for ursolic acid, 100–500 ng for lupeol, and 500–2500 ng per spot for stigmasterol with respect to area. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for oleanolic acid, ursolic acid, lupeol, and stigmasterol were 98.77 to 99.12%, indicating the good reproducibility. Stigmasterol 1.19 ± 0.04% w/w was present at high concentration, and oleanolic acid 0.012 ± 0.006% w/w was present at low concentration in the whole plant powder. The proposed HPTLC method was found to be simple, precise, sensitive, accurate, reproducible, and robust.

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Among the complex mixture of biologically active compounds in Leptadenia pyrotechnica, three compounds have been used as analytical markers. A sensitive high-performance thin-layer chromatographic (HPTLC) method has been developed for the estimation. Methanolic extracts of whole plants from three populations were used on aluminum pre-coated silica gel 60 F254 plates with different mobile phases to determine the amount of β-sitosterol, lupeol, and oleanolic acid with R F value of 0.64, 0.84, and 0.47, respectively. The calibration curve was linear in the range of 2–10 μg. The method is reliable for the quantification, separation, and good resolution of these compounds from other constituents of L. pyrotechnica. To ascertain the purity of the peak from the test sample, its in-situ reflectance spectrum was compared with that from standards; the clear superimposability indicated the purity of the peaks.

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This article enfolds a rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method for the estimation of four triterpenoids, namely, betulin (BU), betulinic acid (BA), lupeol (LU), and oleanolic acid (OA), from the bark, roots, and leaves of Betula utilis D. Don, an endangered Himalayan tree. All the four phytoconstituents have high therapeutic value. Separation was performed on thin-layer chromatography (TLC) aluminum plates precoated with silica 60 F254 (20 × 20 cm) followed by detection of betulin, lupeol, and oleanolic acid carried out by derivatizing the plate with ceric ammonium sulfate followed by heating at 110°C for 5 min. For betulinic acid, the plate was dried and visualized after spraying with Liebermann‒Burchard reagent. CAMAG TLC Scanner 4 equipped with winCATS software was used for densitometric scanning at 500–550 nm. The proposed technique was further validated in terms of linearity, precision, accuracy, and sensitivity as per the International Conference on Harmonisation (ICH) guidelines. A good linear relationship was obtained for the calibration plots with r 2 = 0.9994, 0.9995, 0.9969, and 0.9998 for betulin, lupeol, oleanolic acid, and betulinic acid, respectively. Accuracy of the method was checked by recovery study conducted at three different levels with the average recovery between 98.9% and 99.3% for all the four markers.

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Triterpenoid betulinic acid (BA) was detected, quantified, and reported for the first time from leaf extract of Achyranthes aspera along with much known oleanolic acid (OA). Extraction was achieved using ultrasonic exposure, and reversed-phase.ultra flow liquid chromatographic (RP.UFLC) technique was employed during investigation. RP.UFLC separation was achieved on a Hibar 250–4.6 mm, 5 μ, Lichrospher 100, C18e column using methanol and water (90:10) as mobile phase with pH adjusted to 5.0 using glacial acetic acid (GAA) in an isocratic mode. The content of BA (0.25 mg g−1 fresh weight [FW]) was ∼75% higher than OA (0.06 mg g−1 FW). These results suggest BA to be the major triterpenoid compared to OA in the leaf of A. aspera. High-performance thin-layer chromatography (HPTLC) separation of the two triterpenic acids (oleanolic and betulinic acid) was also achieved on silica gel G 60 F254, 50 ×~ 100 mm glass TLC plates, using benzene, ethyl acetate, and formic acid as solvent system in a ratio of 67.9:22.7:9.4. Anisaldehyde reagent was used for detection. The method was used for the screening of oleanolic and betulinic acids from A. aspera leaf extract. Similarly, Fourier transform-infrared (FT-IR) spectroscopic analysis was done in the mid IR region of 400–4000 cm−1 with 64 scan speed using OMNIC 8.1 (ver. 8.1.210) software. The results were also supported by HPTLC and FT-IR data.

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OA Oleanolic acid IR Insulin resistant HepG2 Human liver tumour BSA Bovine serum albumin AGEs Advanced glycation end products PA Palmitic acid ROS Reactive oxygen species DCFH-DA 2′,7′-dichlorodihydrofluorescein diacetate DM Diabetes AG Aminoguanidine

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Summary

An ecofriendly solvent polarity based microwave-assisted extraction (MAE) technique was developed for the rapid extraction and isolation of bioactive oleanolic acid from roots of Lantana camara L. Several different influential extraction parameters such as microwave power, extraction time, solvent type, and volume were studied in a systematic fashion for the determination of optimum extraction conditions. Simply modified and rapid high-performance liquid chromatography-diode array detector (HPLC-DAD) method was also developed and validated for quantitative determination of oleanolic acid from roots of L. camara. Under optimum conditions, using a mixture of CHCl3:MeOH (60:40, v/v, 15 mL) as a solvent, 600 W microwave powers, and 50 °C temperature for 6 min of MAE produced a maximum yield of 1.23% (dry weight of roots). No degradation of the target analyte was observed at the optimum conditions as evidenced from the recovery studies performed with standard oleanolic acid. The proposed method also showed high degree of reproducibility; hence, it may be useful for maximum extraction and isolation of biologically active oleanolic acid.

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Bark of Myrica esculenta Buch.-Ham., commonly known as ‘Kaphal’ in indigenous systems of medicine, is used for treatment of a variety of ailments. Three samples were collected from the Nainital, Ranikhet, and Shimla regions of India for qualitative and quantitative standardization by HPTLC on silica gel plates. The profiles obtained by use of two mobile phases revealed the presence of almost similar components. Amethod was established for identification of the biomarkers gallic acid, lupeol, oleanolic acid, and stigmasterol in extracts of the plant, because of the therapeutic importance of these compounds. The characteristic band of gallic acid occurred at R F 0.56 when toluene-ethyl acetate-formic acid 5:5:1 was used as mobile phase. Bands of oleanolic acid, stigmasterol, and lupeol occurred at R F 0.38, 0.49, and 0.62, respectively, when toluene-ethyl acetate 8:2 was used. These biomarkers were also estimated quantitatively. The amounts of the biomarkers varies from sample to sample - stigmasterol from 0.2229–0.4489%, oleanolic acid from 0.0079–0.0384%, lupeol from 0.0233–0.0783%, and gallic acid from 0.0795–0.0918%. The amounts were highest in the Nainital sample and lowest in the Shimla sample.

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