Quality assessment and liriodenine quantification of Nelumbo nucifera dried leaf in Thailand

PHCOG J ORIGINAL ARTICLE Quality assessment and liriodenine quantification of Nelumbo nucifera dried leaf in Thailand Saranthinee Mongkolrat 1, Chanida Palanuvej 1* and Nijsiri Ruangrungsi 1,2 1 College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Submission Date: 14-6-2012 ABSTRACT Introduction: Nelumbo nucifera Gaertn. dried leaves are used as crude drug in various Thai traditional recipe. Previous studies found that the leaf extracts exerted potent antioxidant effects, antispasm, direct vasodilatation action and moderate hypotensive effect on experimental animals. Liriodenine is an alkaloid prominent found in Nelumbonaceae. Its anticancer potential has been previously reported. Materials and Methods: N. nucifera leaves from 15 different locations throughout Thailand were examined for pharmacognostic specification. Liriodenine quantification by Thin Layer Chromatography (TLC) image analysis was developed and compared to High Performance Liquid Chromatography (HPLC). Results: Anatomical and histological observation showed air chamber, trichosclereid and calcium oxalate. Pharmacognostic parameters revealed that the acid-insoluble ash, total ash, loss on drying and water content should be not more than 2.61, 9.62, 7.69 and 7.06% of dry weight respectively; while ethanol and water-soluble extractive should be not less than 6.24 and 9.51% of dry weight respectively. TLC image demonstrated clearly fluorescent spot of liriodenine at R f 0.75 under UV 365 nm. Liriodenine content was 4.8 mg% by both methods. Conclusion: This study provided pharmacognostic specification toward fundamental standardization of N. nucifera leaf crude drug in Thailand. Additionally, the simple TLC with image analysis was shown as HPLC alternative to quantify the content of medicinal potential liriodenine alkaloid in this crude drug. Keywords: Liriodenine, Medicinal plant, Quality control, Standardization INTRODUCTION Medicinal plants have been used extensively for treatment since ancient time and became the principle source of medicines at present. Meanwhile Thailand has strong potential for therapeutic application; since 2004 the government has been implemented and developed Thai herbal products to meet international standard. However, adulteration of crude drugs still occurs. Thus to control the quality of medicinal plants, establishing the standard specification is needed. *Address for correspondence. Tel.: +66-2218-8158; fax: +66-2255-2177 E-mail: chanida.p@chula.ac.th DOI: 10.5530/pj.2012.32.5 Nelumbo nucifera Gaertn. (Nelumbonaceae), commonly known in Thai as Bua-luang, is native to tropical Asia and Queensland, Australia. For centuries, sacred lotus is particularly valued for Thai medicinal properties and Thais treat lotus plants as part of their repertoire of medicinal herbs. Virtually all parts of versatile plant including stamens, pollens, flowers, stems, tubers and leaves are used. The leaf part of N. nucifera has been long used as traditional medicine in Asia, including Thailand. The leaf is bitter, sweet and neutral. [1] Previous studies revealed that the leaf extract showed direct vaso-dilatation action, antiinflammation, antioxidation and moderate hypotensive effect on experimental animals. [2 4] Alkaloid are the main products in N. nucifera leaf and have been used as effective drugs for hematemesis, epistaxis and hemoptysis. [5] Moreover, recent studies revealed the effect of alkaloids in the leaf can lower the body weight, the lee s index, adipose tissue weight, and plasma lipid levels in high 24 Phcog J Nov Dec 2012 Vol 4 Issue 32

Figure 1. Chemical structure of liriodenine. fat diet-induced obese rats. [6] Otherwise, large circular leaves are used for food preparation, sometimes applied to be food containers and umbrella. In traditional medicine, it has been combined with any other herbs to treat sunstroke, fever, diarrhea, dizziness and stomach problems. [7] Liriodenine (Figure 1), an aporphine isoquinoline alkaloid constituted in lotus leaves, [8] was selected as a marker in present study according to its interesting properties and many biological activities found including anti-platelet, anti-fungal and anti-microbial actions. [9,10] Additionally recent studies revealed that it had potent cytotoxicity against a number of cancer cell lines. [11] The present study aimed to provide pharmacognostic parameters available for standardization of N. nucifera leaf crude drug in Thailand as well as estimate the liriodenine content of N. nucifera crude drug using TLC image analysis by Scion Image software compared to HPLC. All measurements were done in triplicate. MATERIALS AND METHODS Plant Collection and Extraction N. nucifera leaves were collected from 15 different locations throughout Thailand. Plant specimens were authenticated by one of the authors (N.R.). The voucher and numbers of specimens were deposited at College of Public Health Sciences, Chulalongkorn University, Thailand. Macroscopic, microscopic and quality evaluations of air-dried specimens were examined according to World Health Organization (WHO) quality control methods for medicinal plant materials as briefly described below: [12,13] Crude extracts were performed with 95% ethanol in a Soxhlet apparatus. Pharmacognostic Investigation Macroscopic examination was identified visually for size, color, and other inspections. Free hand sections of the leaf and crude drug powders were performed with a magnification of 4x, 10x, 20x and 40x under microscopy and compared the scale with the 0.01 mm micrometer. Five grams of sample were heated till constantly weight for determination of loss on drying. Then ignited gradually increasing the heat to 500 600 C until it was white, cooled and weighted to calculate total ash. The ash was boiled with 25.0 ml of hydrochloric acid (70 g/l); insoluble matter was ignited and cooled again to constantly weight to yield acid-insoluble ash. Water content was conducted by azeotropic distillation. Determination of extractive values was carried out with 95% ethanol and distilled water as solvents. Clevenger apparatus was applied to determine volatile oil content in ground sample. TLC fingerprinting was performed as follow: the extracted sample was dissolved in methanol; then applied 5 µl to a pre-coated silica gel 60 F 254 plate (Merck, Germany; 0.25 mm thickness, 20 20 cm). The chromatogram was developed in the saturated TLC chamber with the specified solvent as chloroform and methanol (95:5). Removed the plate; allowed it to dry in air and observed the produced spots in daylight, under short wave and long wave ultraviolet light (λ 254 and 365 nm respectively) and sprayed the spots with anisaldehyde staining reagent. Liriodenine Quantification Standard liriodenine (LC/MS grade) was purchased from Specs, The Netherlands. Its purity was reconfirmed by NMR spectroscopy. TLC Image Analysis TLC separation was performed as aforementioned with some modification. After applying 2 µl of each solution onto TLC plates, they were firstly developed using methanol to a distance of 6 mm to expand the band length. [14] Ensuring air-drying, the plates were developed with mobile phase using chloroform and methanol (95:5) to a distance of 80 mm, then dried and visualized under UV 365 nm. The image was taken by digital camera (Canon PowerShot A650). TLC images saved as TIFF files were analyzed by Scion Image program for Windows (version Alpha 4.0.3.2, Scion Corporation, Maryland, USA). After importing images, they were resized with scale to fit windows mode and modified grayscale selection with smoothing menu to reduce noises of image. Using the rectangular selection tool and load macros command to create the plot profile, then the areas and gray value of the selection were measured as square pixels. [15] HPLC Analysis Instrumentation was performed with a SHIMADZU gradient system (Kyoto, Japan) equipped with LC-20AD pumps, a CTO-20AC column oven, DGU-20A3 degasser and a Phcog J Nov Dec 2012 Vol 4 Issue 32 25

Saranthinee Mongkolrat, et al.: Quality assessment and liriodenine quantification of Nelumbo nucifera dried leaf in Thailand SPD-M20A diode array detector (DAD) set λ at 407 nm. Separation was carried out with an Inersil ODS-3, C-18 column (particle size of the packing 5 µm, 4.6 250 mm) and HPLC guard column (5 µm, 4.0 10 mm). The mobile phase consisted of A (formate buffer consisting of 1% formic acid, adjusted to ph 4.5 with diethylamine) and B (100% acetonitrile) from 60:40 to 40:60 over 35 minutes, filtered and degassed with ultrasonic bath prior to use. Each solution was filtered through 0.45 µm syringe filter. Aliquot (20 µl) of each sample was injected into the system at flow rate of 1 ml/min with column temperature set at 25ºC. Calibration Curve and Method Validation According to TLC image analysis, 2 µl of 5 200 μg/ml standard liriodenine dissolved in methanol was applied on TLC plate to generate calibration curve. The standard curve was analyzed using the least square regression equation derived from peak area. In addition standard solutions for HPLC analysis were also prepared in methanol to provide serial concentrations within range 5 200 µg/ml. Linear regression calculated from the peak area was used to construct the calibration curve. Precision, accuracy, limit of detection (LOD) and limit of quantitation (LOQ) were determined using spike method. Figure 2. Whole plant of Nelumbo nucifera. RESULTS AND DISCUSSION N. nucifera is an aquatic perennial herb that grows in ponds, pools, rivers and lakes. The plant grows from a rhizome constricted at its nodes and it s somewhat pinkish. The petiole is to 2 feet long, terete, fistulous, and glabrous. The blade is 25 90 cm in diameter, round, thin, glabrous, and entire at the margin. It floats on the top of water surface. The flowers are conspicuous, 10 25 cm in diameter, pink or white, the petals oblong-elliptic to obovate, 5 cm 11 cm 2.5 cm 5 cm. The fruits are conical, green, and up to 15 cm long[16] (Figure 2). Figure 3 showed greenish brown to brown color in dried leaves. The anatomical and histological investigation of N. nucifera dried leaves demonstrated in Figures 4, 5. Dominant characters from microscopic study demonstrated air chambers, calcium oxalate and trichosclereid on transverse section of leaf and powders. Plant description: The plant grows from a rhizome constricted at its nodes. The petiole is to 2 feet long, terete, fistulous, and glabrous. The blade is 25 90 cm in diameter, round, thin, glabrous, and entire at the margin. It floats on the top of water surface. The flowers are conspicuous, 10 25 cm in diameter, pink or white, the petals oblong-elliptic to obovate, 5 cm 11 cm 2.5 cm 5 cm. The fruits are conical, green, and up to 15 cm long.[16] 26 2 cm Figure 3. Dried leaves of Nelumbo nucifera. Figure 4. Transverse section of Nelumbo nucifera leaf: 1. air chamber 2. trichosclereid 3. calcium oxalate 4. xylem vessle 5. collenchyma 6. upper epidermis 7. trichome 8. palisade cell 9. parenchyma 10. lower epidermis. Phcog J Nov Dec 2012 Vol 4 Issue 32

Figure 5. Powdered Nelumbo nucifera leaves: 1. reticulate vessels 2. tricho-sclereids 3. epidermal cells 4. fragments of parenchyma, longitudinal view 5. vein 6. fibers 7. collenchymas 8. vessels 9. calcium oxalate crystals. Table 1 showed pharmacognostic parameters of N. nucifera dried leaves from 15 different locations throughout Thailand. The acid-insoluble ash, total ash, loss on drying and water content were identified not more than 2.61, 9.62, 7.69 and 7.06% w/w respectively. The determination of ethanol and water-soluble extractive were not less than 6.24 and 9.51% w/w respectively, whereas there was no volatile oil contained in this crude drug. TLC fingerprint and HPLC chromatogram were demonstrated in Figures 6, 7; TLC image showed a well-defined fluorescent spot of liriodenine at the R f value of 0.75 under UV 365 nm. The yields of ethanolic extracts ranged from 15.79 to 34.67% w/w (average of 24.47% w/w). Determination of liriodenine content in dried weight of N. nucifera leaves examined by Scion Image ranged from 0.925 10.971 mg% (average of 4.851 mg%). Then the result was reconfirmed by HPLC analysis that found same average content of liriodenine when compared to TLC image software. There was statistically correlation between both quantitative methods (r = 0.993, p < 0.01). The researcher found that liriodenine could be detected Table 1. Physicochemical Characteristics of N. nucifera Leaves Method Mean ± SD* Min Max Unit Acid-insoluble ashes 2.61 ± 0.24 1.62 3.77 g/100 g Total ash 9.62 ± 0.07 7.94 11.64 g/100 g Loss on drying 7.69 ± 0.07 4.22 10.02 g/100 g Water content 7.06 ± 0.11 4.60 9.02 g/100 g Ethanol-soluble extractive 6.24 ± 0.22 3.37 13.33 g/100 g Water-soluble extractive 9.51 ± 0.24 5.24 12.92 g/100 g Liriodenine content 4.88 ± 2.98 0.93 10.35 mg/100 g (by Scion) Liriodenine content (by HPLC) 4.85 ± 3.10 0.92 10.97 mg/100 g * The parameters were shown as grand mean ± pooled SD. Samples were from 15 different sources throughout Thailand. Each sample was performed in triplicate. Figure 6. TLC fingerprinting of a methanolic extract of dried Nelumbo nucifera leaf: I) Detection under UV light 254 II) Detection under UV light 365 III) Detection with anisaldehyde spraying and heat under visible light IV) Standard liriodenine under UV light 365. Figure 7. HPLC chromatogram of N. nucifera leaf (source no.15); sample concentration was 0.1 g/ml of crude material. Liriodenine peak was at 11 min of retention time, analyzed using SHIMADZU gradient system with Inersil ODS-3, C-18 column (4.6 250 mm), DAD detector at 407 nm. Mobile phase consisted of A (formate buffer consisting 1% formic acid, adjusted to ph 4.5 diethylamine) and B (100% acetonitrile) from 60:40 to 40:60 over 35 min. at 25ºc, flow rate 1 ml/min. under both short and long wavelenghts. Additionally, the compound showed the fluorescent coloring spot on plate that suitably to determine liriodenine content by Scion Image from methanolic extracts of N. nucifera leaves. (Figure 8) Thus, the results illustrated that TLC can be used for quantitative analysis of liriodenine. TLC seperation of crude extracts and visualization by UV light offers a rapid method for the routine and determintion of liriodenine. The procedures described are ideal for low budget research projects and the results obtained can be complemented and confirmed by more complex techniques such as high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). [17] Referring to Liang et al study, the results Phcog J Nov Dec 2012 Vol 4 Issue 32 27

ACKNOWLEDGEMENT Figure 8. TLC image analysis by Scion Image software: A) Importing TLC image saved as TIFF file B) Converting to grayscale and smoothing image C) Chromatograms obtained from plotting profile (From left to right lane: standard liriodenine 5, 25, 50, 100, 200 µg/ml.) indicated that the content of alkaloids, including liriodenine, by HPLC-UV analysis in Zanthoxylum nitidum varied significantly from habitat to habitat. [18] Therefore, variation of liriodenine content may be due to various factors such as effect of growth period and environment, maturity of plant and storage time. Calibration curve of standard compound showed good linearity relationship for both methods (R 2 > 0.995) over the range 5 200 µg/ml. According to TLC image analysis developed, method validation was conducted [19] indicating polynomial regression equation y = 0.1573x 2 + 61.392x + 151.38 where y is AUC and x is concentration. Method gave adequate precision estimated as %RSD for within-day and day-to-day. Limit of detection and quantitation were 0.03 and 0.1 µg/ml, respectively. Accuracy was indicated by the percentage mean of recovery values were 101.12, 101.97, 103.90% of 3 levels of liriodenine addition. CONCLUSION These pharmacognostic investigations could be set as the standard parameter to be useful for quality control and authentication of the N. nucifera leaves crude drug in Thailand. TLC visualization under UV 365 nm with image analysis software and visible detector HPLC method can be applied to quantitatively determine liriodenine containing in plant materials. A B C The authors wish to thank the Herbal Remedies and Alternative Medicine Task Force of STAR: Special Task Force for Activating Research under 100 years Chulalongkorn University Fund. REFERENCES 1. Bensky D, Clavey S, Stoger E. Materis Medica. Eastland Press Inc., USA 2004: 193 4. 2. Pongboonrod S. Exotic Plant in Thailand-Exotic and Thai Medicinal Properties, Bangkok: Krung Thon (1979). 3. Lizhong L, Guohui L. Influence of neferine on proliferation of hypertrophic scar fibroblasts in vitro. Chin. Tradit. Herb. Drugs 2002; 1018 20. 4. Qian JQ. Cardiovascular Pharmacological Effects of Bisbenzylisoquinoline Alkaloid Derivatives. Acta Phamacol Sin 2002; 23(12): 1086 92. 5. Ou M. Chinese English manual of common-used in traditional Chinese MEDICINE. Joint Publishing (H.K.) Co., Ltd., Hong Kong. 1989: 518 19. 6. Bin X, Jin W, Wenqing W, Chunyang S, Xiaolong H, Jiaoguo F. Nelumbo nucifera alkaloid inhibits 3T3-L1 preadipocyte differentiation and improves high-fat diet-induced obesity and body fat accumulation in rats. Journal of medicinal plants research 2011; 5(10): 2021 28. 7. Usha RP. Purslane-Lotus Available source: http://www.lokvani. com/lokvani/article.php?article_id=1036 February, 2008. 8. Wang HM, Yang WL, Yang SC, Chen CY. Chemical constituents from the leaves of Nelumbo nucifera Geartn. cv. Rosa-plena. Chemistry of natural compounds 2011; 47(2): 316 17. 9. Dong XP, Modranodra IO, Che CT. Kmeriol and other aromatic constituents of Kmeria duperreana. Pharmacol Res 1989; 6: 637 40. 10. Clark AM, Watson ES, Ashfaq MK. In vivo efficacy of anti-fungal oxoaporphine alkaloids in experimental disseminated candidiasis. Pharm Res 1987; 4: 495 8. 11. Chang H, Chang F, Wu Y. Anti-cancer effect of liriodenine on human lung cancer cells. Kaohsiung J Med Sci 2004; 20(8): 365 9. 12. World Health Organization. Quality control methods for medicinal plant materials. (NLM Classification QV 766). (1998). WHO, Geneva. 13. Pharmacognostic specification of thai crude drugs. ISBN 978 974 8075 95 2. (2007). 14. Sotanaphun U, Phattanawasin P, Sripong L. Application of Scion Image software to the simultaneous determination of curcuminoids in turmeric (Curcuma longa). Phytochem. Anal. 2008; 20: 19 23. 15. Scion Corporation. Scion Image for Windows. Maryland, USA. 16. Wiart C. Medicinal Plants of Asia and the Pacific. CRC Press 33 39. ISBN 978-0-8493-7245-2. (2006). 17. Mabinya LV, Mafunga T, Brand JM. Determination of ferulic acid and related compounds by thin layer chromatography. African Journal of Biotechnology 2006; 5(13): 1271 3. 18. Liang M, Zhang W, Hu J, Liu R, Zhang C. Simultaneous analysis of alkaloids from Zanthoxylum nitidum by high performance liquid chromatography-diode array detector-electrospray tandem mass spectrometry. Journal of Pharmaceutical and Biomedical analysis 2006; 42: 178 83. 19. ICH International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Validation of Analytical Procedures: Text and Methodology Q2(R1). 28 Phcog J Nov Dec 2012 Vol 4 Issue 32