Lupeol Content Research Articles

Validated high performance thin layer chromatographic method for simultaneous quantification of betulinic acid, β-sitosterol and lupeol in fruits, leaves, root bark and stem bark of Dillenia indica Linn

AbstractDillenia indica is one of the important medicinal plants and is extremely popular in many systems of medicine including ayurvedic, homeopathic, and siddha. Traditionally, different parts of this plant have been used to treat cancer, wound healing, diabetes, diarrhea, bone fractures, abdominal pains, cuts and burns. In the present study, a simple, precise, accurate and validated high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of three pharmaceutically active compounds i.e., betulinic acid (BE), β-sitosterol (BT) and lupeol (LP) in fruits, leaves, root bark and stem bark of D. indica. Standards and plant samples were applied on TLC aluminum plate precoated with 0.2 mm layer of silica gel 60F254. The plate was run in a twin glass chamber comprising toluene:methanol:chloroform (8:1:1, v/v/v) as a mobile phase which resulted in better separation of compounds. The plates were immersed in anisaldehyde-sulfuric reagent and then heated at 105 °C for 5 min in CAMAG TLC plate heater for appearance of bands. Densitometric scanning was performed at λmax = 525 nm using tungsten light source in CAMAG TLC Scanner4 armed with WinCATS software. RF values of BE, BT and LP standards and those of plant samples were found to be 0.38 ± 0.01, 0.54 ± 0.01 and 0.65 ± 0.02 respectively. The method was further validated by following the International Conference of Harmonization (ICH) guidelines. For BE, BT and LP, the linear regression data for the calibration plots revealed a satisfactory linear association with correlation coefficients (r2) = 0.9736, 0.9989 and 0.9957, respectively. Linear ranges for BE, BT and LP were 2,000–6,000 ng/band, 200–1,000 ng/band, and 200–600 ng/band respectively. Accuracy of the method was assessed by recovery study conducted at three different levels with the average recovery of 99.19, 99.69 and 100.95% for BE, BT and LP, respectively. The results exhibited the highest content of BE (0.148 ± 0.01%), BT (0.031 ± 0.01%) and LP (0.047 ± 0.01%) in stem bark. The developed method will be useful for routine and quality control analysis of fruits, leaves, root bark and stem bark of D. indica species.

Phytochemical Marker-based Analysis, Antioxidant Potential, and Shelf-Life Study of Geographically Different Samples of Tinospora cordifolia (Stem)

Background: Tinospora cordifolia (TC), commonly referred to as Guduchi, holds a prominent position in the Indian traditional system of medicine and has been cited in Ayurvedic literature for its efficacy in the treatment of a range of ailments, including jaundice, urinary issues, diabetes, prolonged diarrhea, skin conditions, stomachaches, and dysentery. Methods: The present study analyzed the content of phytochemical markers present in the stems of TC collected from five distinct geographical locations (TC-01 to TC-05) and assessed the corresponding variations in their in-vitro antioxidant potential. Results: Lupeol content in various samples of TC varied from 217.9 to 510.9 μg/mg, while palmatine ranged from 0.002% to 0.025%. Total phenolic content (TPC) ranged from 0.77 to 6.35 mg GAE/g wt., and total flavonoid content (TFC) from 12.15 to 20.03 mg QCE/g wt., with IC50 values between 21.02 and 81.81 mg/mL. Elemental analysis of TC-01 to TC-05 showed significant essential elements and permissible levels of heavy metals. A shelf-life study of TC samples indicated stability beyond three years. Conclusion: The considerable diversity in phytochemical content and antioxidant values underscores the substantial influence of geographical variation on the therapeutic potential of TC.

Spectral characterization, analgesic, and anti-inflammatory effects of ethanolic extract of Calotropis procera leaf and dry latex from Jazan, Kingdom of Saudi Arabia

Traditional healers have used the shrub Calotropis procera (CP) for many years for various therapies. The present study investigated the bioactive constituents of ethanolic extract of CP leaf and dried latex using gas chromatography-mass spectrometry and Fourier transforms infrared spectroscopy. The identification and characterization of the compounds were confirmed by examining the constituents' mass spectrum fragmentations and FT-IR spectra and comparing the results with those in the literature. The tail-flick method was used to investigate the analgesic properties of the extract and its anti-inflammatory activities using a rat model of formalin-induced oedema. Acute oral toxicity in rats was studied per OECD recommendations. Twenty male rats were divided into four groups and received an ethanolic extract of the leaves and dried milky sap of CP (200 mg/Kg) in groups 1, 2, and 3. Group 4 rats were administered aspirin 50 mg/kg as a positive control. The CP dried latex extract has the highest content of lupeol and its acetate derivative compared to its leaf extract. The CP dried latex extract inhibited inflammation more significantly than the ethanolic leaf extract and the drug indomethacin at a higher dosage (200 mg/kg). The ethanolic extracts showed analgesia comparable to aspirin. It suggests that fatty acids and their esters, particularly ethyl linoleate (8.96%), ethyl palmitate (7.99%), ethyl linoleate (6.98%), and palmitic acid (5.18%), may be valuable biomarkers for characterizing leaf and latex samples and describing the medicinal potential of CP.

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus.

Cytochrome P450 monooxygenases (CYPs) are enzymes that play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations of the triterpene scaffold, CYPs require electrons transferred by NADPH-cytochrome P450 reductase (CPR), which is classified into two main classes, class I and class II, based on their structural difference. Lotus japonicus is a triterpenoids-producing model legume with one CPR class I gene (LjCPR1) and a minimum of two CPR class II genes (LjCPR2-1 and LjCPR2-2). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of L. japonicus hairy root culture treated with methyl jasmonate (MeJA), this study revealed that LjCPR1, CYP716A51, and LUS were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, LjCPR2s, bAS, CYP93E1, and CYP72A61 were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. β-amyrin, 24-OH β-amyrin, and sophoradiol content. Triterpenoids profile analysis of LORE1 insertion and hairy root mutants showed that the loss of the Ljcpr2-1 gene significantly reduced soyasaponins precursors but not in Ljcpr1 mutants. However, Ljcpr1 and Ljcpr2-1 mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, LjCPR1, but not LjCPR2, was crucial for seed development, supporting the previous notion that CPR class I might support plant basal metabolism. This study suggests that CPR classes I and II play different roles in L. japonicus triterpenoid biosynthesis.

Elicitor mediated enhancement of shoot biomass and lupeol production in Hemidesmus indicus (L.) R. Br. ex. Schult. and Tylophora indica (Burm. F.) Merrill using yeast extract and salicylic acid

Hemidesmus indicus (L.) R. Br. ex Schult. and Tylophora indica (Burm. F.) Merrill shoot cultures were treated with different concentrations of yeast extract (YE; 25–200 mg/L) and salicylic acid (SA; 50–200 µM), and their effect on lupeol production was assessed. The maximum dry weight (DW) biomass was recorded when H. indicus shoots were treated with SA (50 µM) and T. indica shoots with YE (200 mg/L). Highest lupeol yield (335.40 ± 0.04 µg/g DW) was obtained in H. indicus shoots after treatment with 50 µM of SA for 3 weeks. Whereas in T. indica, maximum lupeol content (584.26 ± 8.14 µg/g DW) was recorded by giving treatment with 25 μM of SA for 6 weeks.

Quantitation of lupeol from stem bark extract of Betula alnoides Buch.-Ham. ex D.Don by two validated RP-HPLC and TLC-densitometric methods

To control the quality of Betula alnoides stem bark (BAS), lupeol, which is a promising compound found in BAS, has been determined by reverse phase high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC)-densitometry. These methods were developed and validated according to the International Council for Harmonization (ICH) and the Association of Official Agricultural Chemists (AOAC) guidelines. RP-HPLC was achieved within 10 min using C8 column under an isocratic elution of acetonitrile at a flow rate of 1.0 mL/min. Lupeol was detected at a UV wavelength of 200 nm. TLC-densitometry performed on TLC silica gel plates was developed by chloroform at 10 cm of distance. Lupeol was detected under visible wavelength at 525 nm after anisaldehyde-sulfuric acid derivatization. Both developed methods exhibited good linearity, specificity, limits of detection and quantitation, accuracy and precision, and robustness. The validated methods were effectively applied to determine lupeol content in BAS collected from three different locations. The amount of lupeol was found to be about 50–100 µg/g of dried powder. The results revealed no statistically significant differences between the two methods. These findings revealed the validated RP-HPLC and TLC-densitometry that were easily applied for routine quality control of BAS for the first time.

Quantitative analysis of triterpene lupeol and anti-inflammatory potential of the extracts of traditional pain-relieving medicinal plants Derris scandens, Albizia procera, and Diospyros rhodocalyx.

ABSTRACTDerris scandens, Albizia procera, and Diospyros rhodocalyx have traditionally been used as herbal remedies for pain relief in Thailand. The ethanolic extracts of these plants obtained by Soxhlet extraction were analyzed by the developed high-performance liquid chromatography-diode-array detection method. Lupeol, the anti-inflammatory triterpene, was selected as a chemical marker for this investigation. All extracts together with that compound were further evaluated for their potential on anti-inflammatory activity using 5-lipoxygenase inhibition assay. Lupeol in each extract was quantified and expressed in the range of 21.44 ± 0.89–40.72 ± 0.40 mg per 100 g of crude drug and the enzyme inhibitory activity of all tested extracts presented as half-maximal inhibitory concentration values ranged between 63.71 ± 2.09 and 91.09 ± 1.40 μg/mL. This study shows that the developed analytical method is effective for analyzing triterpene lupeol in these plants and also reveals the relationship between a lupeol content and the anti-inflammatory effect.

Evaluation of the in vitro biological activities of Banana flower and bract extracts and their bioactive compounds

ABSTRACT In this study, the banana flower was divided into bract and male flowers and extracted with water and ethanol, at different temperatures, and then the functionality of the obtained extract was evaluated and the bioactive compounds were analyzed. The findings showed that the banana flower with an ethanol extract of 50% and a temperature of 50°C (FE-50%-50°C) had the best DPPH radical scavenging ability and hydroxyl radical scavenging ability of 97.44% and 38.86%, respectively. Moreover, the FE-50%-50°Calso had the best reducing power. In terms of its ferrous ion chelating ability, the banana flower ethanol 50% extract at room temperature (FE-50%-RT) had the best ferrous ion chelating ability of 96.82%. The 95% ethanol extract of the banana flower was effective in inhibiting α-amylase, α-glucosidase, and lipase activity was 31.75%, 84.17%, and 70.59%, respectively. FE-50%-50°C had the highest alcohol dehydrogenase activity of 74.92%. In addition, the FE-95%-50°C had the highest content of lupeol and umbelliferone, which were 40.10 and 90.83 μg/mg, respectively.

Impact of seasonal variation on lupeol content in latex of Calotropis gigantea R.Br. and Calotropis procera Ait.

Impact of seasonal variation on lupeol content in latex of Calotropis gigantea R.Br. and Calotropis procera Ait.

Impact of seasonal variation on lupeol content in latex of Calotropis gigantea R.Br. and Calotropis procera Ait.

Impact of seasonal variation on lupeol content in latex of Calotropis gigantea R.Br. and Calotropis procera Ait.

Regeneration and chemical profiling in Hemidesmus indicus (L.) R. Br.

Hemidesmus indicus (L.) R. Br. (Anantmoola) is an important medicinal plant of India, which is being used in many pharmaceutical as well as ayurvedic preparations. The present study deals with the development of an efficient rapid shoot regeneration along with their biosynthetic potential. Shoot cultures were established from nodes of H. indicus using Murashige and Skoog's (MS) medium fortified with sucrose (3%), different concentrations and combinations of cytokinins viz. benzyladenine (BA) and kinetin (Kn), auxins viz. indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA). Optimum number of shoots (11.00±0.24 shoots/explant) were observed in MS medium supplemented with combination of BA (10μM) and Kn (5μM). These shoots were subcultured to IAA (2μM) supplemented medium for another eight weeks, and shoots grown in cytokinin [BA (10μM) and Kn (5μM)] and auxin [IAA (2μM)] based media were analyzed for their biosynthetic potential using high performance thin layer chromatography (HPTLC). Chemical fingerprinting and lupeol quantification was carried out to check biosynthetic potential. The fingerprints of the wild and in vitro samples showed similar bands in cytokinin fortified medium whereas variation in chemical constituents was observed in terms of number of peaks and band on HPTLC plate in shoots from auxin fortified medium. Maximum lupeol content (0.187±0.01mg/g) was estimated in shoots regenerated in BA (10μM) and Kn (5μM) which was slightly higher than wild shoots (0.185±0.00mg/g). This protocol can be utilized for mass propagation, conservation and production of lupeol.

Pharmacognostic Standardization and Chromatographic Fingerprint Analysis on Triterpenoids Constituents of the Medicinally Important Plant Plumeria rubra f. rubra by HPTLC technique

Introduction: Plumeria rubra f. rubra commonly known as Lal Gulachin has wide horizon of medicinal possessions. Plant is found in India and in its tropical regions. Though the plant and its extracts have been indigenously valued as folklore medicine diversely in India, yet literature lacks somewhere in reverse pharmacognostical approach of this plant which reflects that plant have not been evidently explored therapeutically. There are several forms of Plumeria rubra among which P. rubra f. rubra is much appraised in India than its other forms. Method: In Present study the anticipated potential of this plant has been validated by laying down its pharmacognostical standards along with measurement of its active therapeutic constituent Ursolic acid and Lupeol via. HPTLC, information from organized search of published literature remarks that Ursolic acid and lupeol is ubiquitous to this plant. Results: Microscopic features revealed the presence of paracytic type of stomata, crescent bicollateral vascular bundle, calcium oxalate crystal and clothing trichomes in leaves whereas bark showed the presence of distinct periderm with cork and phellogen, sclereids, bast tissue with parenchymatous cells. Methanolic extract of both parts of plant was subjected to HPTLC. In HPTLC studies the Ursolic acid content in leaves was found to be 0.96% whereas in bark was detected as 0.051%, lupeol content in leaves and stem was found to be 0.014% and 0.018%. Conclusion: The data generated could be significantly used as reference for the standardization and quality control of Plumeria. rubra f. rubra, as no such work has been reported yet.

Comparative Assessment of Lupeol in Wild plant and In Vitro Callus Culture of Hemidesmus indicus by HPLC Technique

Medicinal plants are used as traditional medicines throughout the world for thousands of years and continue to provide new remedies to mankind. Plants are one of the richest sources of secondary metabolites. The roots of Hemidesmus indicus served as the remedy for leprosy, syphilis, leucoderma, asthma, dysentery, fever and blood, kidney and urinary diseases. The aim of the present study was to determine the quantity of lupeol present in callus and wild plant extract of Hemidesmus indicus. The root callus was initiated on MS medium fortified with varied concentrations and combination of different auxins like, 2-4 Dichloro acetic acids (2-4D), napthalene acetic acid (NAA) and cytokinin like, kinetin (Kn) in 250 ml culture flasks. The content of lupeol in callus and root extracts was analyzed by HPLC technique. The highest percentage of callus induction (88.00 %) was observed in MS medium (C7) containing 1.0 mg/l NAA with highest callus growth in terms of fresh weight (946.48±6.6) which resulted in soft friable callus. The callus produced by C7 was used selected for further study. The HPLC study revealed that the quantity of lupeol present in callus extract was more compared to the wild plant extract of Hemidesmus indicus.

Uni-dimensional double development HPTLC-densitometry method for simultaneous analysis of mangiferin and lupeol content in mango (Mangifera indica) pulp and peel during storage

Mango (Mangifera indica) fruit is one of the important commercial fruit crops of India. Similar to other tropical fruits it is also highly perishable in nature. During storage/ripening, changes in its physico-chemical quality parameters viz. firmness, titrable acidity, total soluble solid content (TSSC), carotenoids content, and other biochemicals are inevitable. A uni-dimensional double-development high-performance thin-layer chromatography (UDDD-HPTLC) method was developed for the real-time monitoring of mangiferin and lupeol in mango pulp and peel during storage. The quantitative determination of both compounds of different classes was achieved by densitometric HPTLC method. Silica gel 60F254 HPTLC plates and two solvent systems viz. toluene/EtOAC/MeOH and EtOAC/MeOH, respectively were used for optimum separation and selective evaluation. Densitometric quantitation of mangiferin was performed at 390nm, while lupeol at 610nm after post chromatographic derivatization. Validated method was used to real-time monitoring of mangiferin and lupeol content during storage in four Indian cultivars, e.g. Bombay green (Bgreen), Dashehari, Langra, and Chausa. Significant correlations (p<0.05) between of acidity and TSSC with mangiferin and lupeol in pulp and peel during storage were also observed.

Constituents of Twig Bark of Pear Cultivars (Pyrus species)

Organic solvent extracts from fresh twig bark of Japanese pear cultivars (Pyrus serotina) Shinko and Nijisseiki, and European pear cultivar (P. communis) Le Lectier were obtained by maceration with n-hexane and EtOAc, and analyzed in GC-EIMS experiments. In these two Japanese cultivars, the lupeol, betulin, epifriedelinol, friedelin and arbutin contents of Nijisseiki were higher than those of Shinko. In the case of the lupane-type triterpenes, lupeol and betulin, the lupeol content of Japanese pears Shinko and Nijisseiki was higher than that of European pear Le Lectier. The betulin content of Le Lectier was higher than those of Shinko and Nijisseiki. Friedelane-type triterpenes, epifriedelinol and friedelin, were not detected in twig bark of Le Lectier. Quantitative and qualitative differences in the constituents of these three pear cultivars were observed.

Comparative Botanical and Phytochemical Evaluation of Medicinally Important Stem Bark of Ficus species

ObjectiveTo evaluate the pharmacognostical comparison (Botanical study, physicochemical parameters, HPTLC analysis) in stem barks of four Ficus species (family-Moraceae) viz. F. religiosa L, F. glomerata Roxb, F. retusa auct. &F. carica. MethodsEstimation of Phytochemical markers viz: β-sitosterol and lupeol was quantified by HPTLC method and antioxidant studies by carried by DPPH method. ResultsHPTLC method showed considerable amount of variation with two reference standard viz: β-sitosterol and lupeol content in stem bark of F. religiosa, F. glomerata, F. retusa &F. carica and it were found 0.084, 0.041, 0.059 & 0.131 and 0.020, 0.043, 0.069 & 0.049 respectively. The antioxidant potential of ethanolic extract of stem bark of F. religiosa, F. retusa, F. glomerata &F. carica were found 46.86%, 42.56%, 31.25% & 25.63% at 0.1mg/mL concentration. ConclusionThe present work was taken up with a view to lay down standards which will contribute significantly to quality control of these medicinally useful Ficus species. It also provides suitable criteria to differentiate the stem barks of four Ficus species.

QUALITY ASSESSMENT FOR PORTULACA OLERACEA BY MULTI-COMPONENT QUANTIFICATION, CHROMATOGRAPHIC FINGERPRINT AND RELATED CHEMOMETRIC ANALYSIS

For the reliable quality control of Portulaca oleracea L., multi-component simultaneous quantification, chromatographic fingerprint, and related chemometric techniques were applied for exploration. In order to obtain the best extraction efficiency and chromatographic resolution, extraction method and chromatographic conditions were optimized. The samples from different origins were collected and identified. With the use of the Ultra Performance Liquid Chromatography (UPLC) and coupled Photo-Diode Array (PDA) detector, 18 characteristic peaks were comprised in the fingerprint common pattern. Among them, four marked components, including hesperidin, kaempferol, lupeol, and myricetin, were quantified. Similarity evaluation, principal component analysis, and hierachical cluster analysis were applied to demonstrate the distinction. All the results implied two conclusions: (1) chemical compositions of P. oleracea from different origins were very different; and (2) the simple, feasible, and effective method for quality control of P. oleracea was to determine the content of lupeol and then make a comparison of fingerprint atlas.

Determination of Lupeol in Fruits by RP-HPLC

A simple method was developed for determining lupeol in fruits by reverse-phase high performance liquid chromatography(RP-HPLC).Separation was carried out on reversed-phase Nucleosil C18 column(5μm,250 mm × 4.6 mm i.d.) by using a mixture of methanol and water(20:80,pH 3.2 ± 0.05 adjusted by 50% phosphoric acid) as mobile phase A,and methanol containing 0.01% phosphoric acid as mobile phase B(A:B = 4:96) at a flow rate of 1.2 mL/min.The column temperature was hold at 40 ℃,and the detection wavelength was set as 210 nm.The correlation coefficient of the linear calibration curve(y = 4.6068x + 11.2640) was 0.9992 at the concentration range of 5 to 250μg/mL.The relative standard deviations(RSDs) of retention time and peak area in precision experiments for 5 replicate analyses and 9 replicate analyses in 48 h were 0.2107% and 1.2843%,and 0.3383% and 1.9737%,respectively.The recovery rate range was between 94.971% and 101.964% with a RSD of 2.5825%.The detection limit of this method was 1.8886μg/mL.The content of lupeol in dried Chinese olive powder was 359 μg/g,which was higher than that in strawberry,banana,Jufeng grape,American red grape and papaya.Therefore,the developed method is practicable for qualitative and quantitative determination of lupeol in fruits.

Production of Dried Extracts of Pterodon emarginatus (Fabaceae) by Atomization

The specie Pterodon emarginatus Vogel is popularly known as „sucupira branca“, which is a native tree from Brazilian savanna (cerrado). This tree is used as wood source for carpentry, while the extract from seeds and barks is popularly used as antinflammatory and antimicrobial for throat or gynecologic infections. The alcoholic extract of stem bark of P. emarginatus exhibited both anti-inflammatory and anti-nociceptive activities [1]. These biological effects can be associated to the presence of great amount of lupeol, which was taken as chemical marker in these experiments to validate our study. In order to evaluate the process for production of P. emarginatus extracts dried by atomization (spray dryer) on a laboratory scale, an experimental factorial design (22) was carried out for the following variables: inlet temperature and air flow. The higher and lower levels for temperature were 100°C and 110°C and for airflow were 35 L/min and 40 L/min. The experimental responses were dried powder yield and lupeol content, which were determined by HPLC. The main effects and interactions of these experimental responses were calculated using the free software Octave. Micrographs obtained by scanning electron microscopy (SEM) of all spray-dried powders were used to examine particle morphology. Experimental results suggest that both temperature and air flow showed a positive effect on yield. However, the interaction between these variable showed a negative effect, which this can be explained by the reduction of the overall yield caused by augmentation of airflow when the experiment was carried out at 110°C. It was observed that there was no significant effect of inlet temperature and airflow on lupeol content. SEM images analysis showed that all powders have a non-uniform morphology. It was observed that particles with different sizes had a tendency to form agglomerates, particularly for the sample obtained at 100°C and 40 L/min. Acknowledgments: The authors are grateful to Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG) for financial support and to Laboratório Nacional de Luz Síncroton (LNLS) for SEM analysis.

An HPTLC Method for the Evaluation of Two Medicinal Plants Commercially Available in the Indian Market Under the Common Trade Name Brahmadandi

Brahmadandi is an important medicinal plant used in the Indian system of medicine (Ayurveda) for the treatment of numerous diseases. A literature search revealed that different plants are available on the market under the trade name Brahmadandi viz., roots of Echinops echinatus Roxb, and the aerial parts of Tricholepis glaberrima DC which are sold either in their crude or in powdered form. Currently, no analytical procedures appear to be available for quality control purposes. In the present communication, we report a simple HPTLC method for the quantification of the lupeol content in the aforementioned plant species. The method was validated for precision, repeatability and accuracy. Instrument precision and repeatability of the method were found to be 0.56 and 2.87 %RSD, respectively. Intra-day and inter-day precision of the method was determined to be in the ranges of 0.71–2.02 and 1.03–2.02 %RSD, respectively. Accuracy of the method was evaluated by a recovery study conducted at three different levels. The mean percentage recovery was found to be 100.85%. The developed HPTLC method for estimation of lupeol was found to be simple, precise and accurate and may be useful for routine quality control of the commercial samples of Brahmadandi.