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MEDICINAL CHEMISTRY | RESEARCH ARTICLE
HPTLC-based quantification of camptothecin in Ophiorrhiza species of the southern Western Ghats in IndiaRenjith Rajan, Sibi Chirakkadamoolayil Varghese, Rajani Kurup, Roja Gopalakrishnan, Ramaswamy Venkataraman, Krishnan Satheeshkumar and Sabulal Baby
Cogent Chemistry (2016), 2: 1275408
Rajan et al., Cogent Chemistry (2016), 2: 1275408http://dx.doi.org/10.1080/23312009.2016.1275408
MEDICINAL CHEMISTRY | RESEARCH ARTICLE
HPTLC-based quantification of camptothecin in Ophiorrhiza species of the southern Western Ghats in IndiaRenjith Rajan1, Sibi Chirakkadamoolayil Varghese2, Rajani Kurup1, Roja Gopalakrishnan3, Ramaswamy Venkataraman4, Krishnan Satheeshkumar2 and Sabulal Baby1*
Abstract: Camptothecin (CPT), a modified monoterpene indole alkaloid, is a po-tential anticancer drug, and due to high demand, search for its new plant-based sources is a priority. Genus Ophiorrhiza is a candidate group in the search for new resources of CPT. Here, CPT contents in 38 Ophiorrhiza accessions, belonging to 11 species and 3 varieties, collected from the southern Western Ghats region in India were quantified by HPTLC-densitometry. Ophiorrhiza mungos (396.54 μg/g, dr. wt.) and O. mungos var. angustifolia (373.19 μg/g, dr. wt.) were the two best CPT sources among the screened species/varieties. O. rugosa var. decumbens (18.55 μg/g, dr. wt.) and O. hirsutula (17.14 μg/g, dr. wt.) showed moderate contents of CPT. This is the first systematic CPT screening of O. hirsutula, O. barnesii, O. incarnata, O. radicans and O. villosa. This study shows the significance of choosing high CPT-yielding eco-types/chemotypes of Ophiorrhiza species or varieties for commercial purposes.
Subjects: Bioscience; Pharmaceutical Science; Pharmacology; Biology
Keywords: Ophiorrhiza species; camptothecin; ecotypes/chemotypes; HPTLC-densitometry
1. IntroductionCamptothecin (CPT) (Figure 1), a topoisomerase inhibitor, was first isolated from Camptotheca acuminata in 1958 (Wall et al., 1966), and it became a potential anticancer drug through a series of
*Corresponding author: Sabulal Baby, Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India E-mails: [email protected], [email protected]
Reviewing editor:Youcef Mehellou, University of Birmingham, UK
Additional information is available at the end of the article
ABOUT THE AUTHORDr Sabulal Baby is Principal Scientist and Head at the Phytochemistry and Phytopharmacology Division of Jawaharlal Nehru Tropical Botanic Garden and Research Institute at Thiruvananthapuram in India. Dr Sabulal received his doctorate in Chemistry from the Indian Institute of Technology Bombay. He did postdoctoral research at the University of Pennsylvania, USA and University of Toronto, Canada. His research areas are phytochemistry and biochemistry. He published over fifty peer reviewed papers in these research areas.
PUBLIC INTEREST STATEMENTCamptothecin (CPT), a modified monoterpene indole alkaloid, is a potential anticancer drug. High demand of CPT as a drug necessitates its supply demand equilibrium, and prompts to search for its new natural sources. Genus Ophiorrhiza is a candidate group in the search for new resources of CPT. A total of 82 accessions (phase I 44, phase II 38) of 14 species and 3 varieties in genus Ophiorrhiza collected from the southern Western Ghats region in India were screened for CPT using HPTLC-densitometry. We found O. mungos and O. mungos var. angustifolia as the two best sources of CPT in genus Ophiorrhiza. Some of the screened Ophiorrhiza species/varieties showed zero or non-detectable levels of CPT. This study shows the significance of choosing high CPT-yielding ecotypes/chemotypes of Ophiorrhiza species or varieties for commercial purposes.
Received: 06 October 2016Accepted: 19 December 2016First Published: 23 December 2016
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© 2017 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license.
Sabulal Baby
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clinical trials (Hsiang, Hertzberg, Hecht, & Liu, 1985; Li, Zu, Shi, & Yao, 2006; Liu et al., 2000; Pommier, 2006). However, low aqueous solubility and toxicity of CPT led to its temporary withdrawal from the market, and semisynthetic analogs replaced it (Khazir, Mir, Pilcher, & Riley, 2014; van Hattum et al., 2002). Newer semisynthetic CPT analogs such as Karenitecin, Diflomotecan, and Gimatecan are in various stages of clinical trials against advanced stages of solid tumors (Garcia-Carbonero & Supko, 2002; Zunino & Pratesi, 2004). Recent clinical studies revealed two other CPT analogs Elomotecan and DRF-1042 as potent drugs against various tumors (Khazir et al., 2014). Natural CPT derivatives 9-methoxy CPT and 10-hydroxy-CPT were also isolated (Lorence & Nessler, 2004), and both them are water soluble with antitumor potentials.
Recent advances in therapeutic strategies using target oriented drug delivery systems made CPT a major candidate in the treatment of multi-drug resistant cancers (Porta et al., 2013; Tang, Cao, & Cheng, 2014). Newer drug delivery with liposomal and copolymer vehicle mediated systems drasti-cally improved the safety and efficacy of CPT (Bissett et al., 2004; Khazir et al., 2014; Wachters et al., 2004). CPT release mediated by pH-sensitive block copolymers was also reported recently (Luo, Yang, Xu, Chen, & Zhao, 2014). Stem cell-based research in apoptogenic signaling of CPT is also in progress (García et al., 2014). This could stimulate CPT-based applications in regenerative medicine. In addition to its basic anticancer properties, CPT is also used for other pharmacological activities (López-Meyer, Nessler, & McKnight, 1994).
CPT has been isolated from various plants such as C. acuminata, C. lowreyana, C. yunnanensis, Nothapodytes nimmoniana, Pyrenacantha klaineana, Merrilliodendron megacarpum, Ervatamia heyneana, Mostuea brunonis, O. mungos, O. pumila, and O. filistipula (Lorence & Nessler, 2004). Due to its demand, the search for alternative, viable plant-based sources of CPT is a priority. Cultivable and tissue culture multipliable plants are the priority sources of CPT. Ophiorrhiza is a candidate group in this resource search for CPT (Renjith et al., 2013). Genus Ophiorrhiza belongs to the family Rubiaceae and is represented by 47 species and 9 varieties in the Indian subcontinent (Deb & Mondal, 2001). Sixteen species and 3 varieties are reported from the southern Western Ghats region in India (Joseph & Joseph, 2009; Sasidharan, 2004). In the first phase of this work, we reported CPT contents in 44 accessions belonging to 9 Ophiorrhiza species, and 3 varieties (Renjith et al., 2013). HPTLC–den-sitometry is one of the techniques useful in chemical profiling of plant extracts and quantification of the bioactive molecules in them (Haridas, Rajani, Mathew, & Sabulal, 2015; Renjith et al., 2013; Thomas et al., 2010). CPT contents in plant sources (C. acuminata) and in their tissue cultured sys-tems were also analyzed by HPLC-based techniques (López-Meyer et al., 1994; Sankar-Thomas & Lieberei, 2011). Here, in the second phase of this screening program, we report the quantification of CPT contents in 38 accessions of Ophiorrhiza, collected from southern Western Ghats, belonging to 11 species and 3 varieties by HPTLC-densitometry.
2. Results and discussionCPT peaks (Rf 0.42 ± 0.01, n = 20) in Ophiorrhiza extracts were well resolved from other signals (Figure 2). CPT contents (Table 1) in Ophiorrhiza extracts were determined by means of the calibra-tion plot, y = (2 × 106 + 06) × + 67.12, R2 = 0.999, and linearity of the calibration curve in the range
Figure 1. Camptothecin.
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0.0001–0.004 μg was ensured. On repeated measurements, mean percentage recovery observed for CPT was 100.10 ± 0.72 (Table 2). % Residual Standard Deviations (RSD) (for 20 accessions, n = 6) were determined as 1.02. Limit of detection (LOD, 25 accessions, n = 6) and limit of quantification (LOQ, 25 accessions, n = 6) were determined for CPT as 0.000085 and 0.000258 μg, respectively.
CPT contents (μg/g, dr. wt.) in 38 accessions of 11 species and 3 varieties of Ophiorrhiza collected from various locations in Western Ghats are given in Table 1. O. mungos (396.54 μg/g, dr. wt.) and O. mungos var. angustifolia (373.19 μg/g, dr. wt.) were the two best sources of CPT among the species/varieties screened. O. mungos (O. mungos var. mungos) is a herb of 8–100 cm height. O. mungos var. angustifolia is a smaller herb of 20–35 cm height, and it is a better candidate for cultivation and tis-sue culture based multiplication (Renjith et al., 2013). O. mungos and O. mungos var. angustifolia showed distinctly different chemical profiles on HPTLC under identical conditions (Renjith et al., 2013; Figure 2), justifying their taxonomic status (Deb & Mondal, 2001). O. rugosa var. decumbens (18.55 μg/g, dr. wt.) and O. hirsutula (17.14 μg/g, dr. wt.) showed moderate CPT contents. Various Ophiorrhiza species (or varieties) showed significant intra and inter species variations in their CPT contents. The present data clearly support our previous report of CPT contents in species/varieties
Figure 2. HPTLC profile of the (a) MeOH extract of O. mungos (52748) collected from Brymore, Thiruvananthapuram in Kerala, (b) CPT standard (0.003 μg/spot).
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Table 1. Estimation of CPT in Ophiorrhiza species/varieties by HPTLC-densitometry
aEach CPT ± SD data is an average of six values. bOphiorrhiza mungos var. mungos (Deb & Mondal, 2001) is commonly termed as Ophiorrhiza mungos (Lorence & Nessler, 2004; Rehman et al., 2009), and thus counted as a species (not as a variety) in our data (Renjith et al., 2013).
Accession No. Species Location District CPT ± SD (μg/g, dr. wt.)a
69998 Ophiorrhiza barnesii Kadalar Idukki 0.00 ± 0.00
52740 O. caudata Thattekadu Ernakulam 0.00 ± 0.00
52722 O. eriantha Chemmunji Thiruvananthapuram 0.00 ± 0.00
69943 O. eriantha Attayar Thiruvananthapuram 0.00 ± 0.00
69982 O. eriantha Pandimotta Kollam 0.06 ± 0.02
52720 O. grandiflora Chummunji Thiruvananthapuram 0.00 ± 0.00
52732 O. hirsutula Periya Wayanadu 17.14 ± 4.42
52733 O. incarnata Periya Wayanadu 0.00 ± 0.00
52727 O. mungosb Silent Valley Palakkad 396.54 ± 0.81
52744 O. mungos Pooyamkutty Ernakulam 354.55 ± 12.95
52748 O. mungos Brymore Thiruvananthapuram 303.32 ± 6.35
52750 O. mungos Idinjar Thiruvananthapuram 276.66 ± 4.53
52751 O. mungos Palode Thiruvananthapuram 227.47 ± 6.29
52753 O. mungos Aluvamkudi Pathanamthitta 148.33 ± 3.20
52755 O. mungos Konni Pathanamthitta 215.37 ± 6.99
69923 O. mungos Kunthirikkapalam Idukki 339.98 ± 28.65
52734 O. mungos var. angustifolia Periya Wayanadu 373.19 ± 0.04
52746 O. mungos var. angustifolia Pooyamkutty Ernakulam 211.07 ± 14.50
69918 O. mungos var. angustifolia Bonacaud Thiruvananthapuram 214.05 ± 5.84
69961 O. mungos var. angustifolia Plappally Pathanamthitta 110.13 ± 7.98
52745 O. pectinata Pooyamkutty Ernakulam 0.09 ± 0.01
52752 O. pectinata Aluvamkudi Pathanamthitta 0.04 ± 0.01
69926 O. pectinata Thekkadi Idukki 2.84 ± 0.02
69935 O. pectinata Anakulam Idukki 0.26 ± 0.01
69936 O. pectinata Anakulam Ernakulam 0.24 ± 0.016
69956 O. pectinata Angamoozhi Pathanamthitta 0.67 ± 0.02
52736 O. radicans Rosemala Thiruvananthapuram 0.00 ± 0.00
52724 O. rugosa var. decumbens Silent Valley Palakkad 18.55 ± 0.02
52725 O. rugosa var. prostrata Silent Valley Palakkad 1.51 ± 0.00
52735 O. rugosa var. prostrata Rosemala Thiruvananthapuram 0.00 ± 0.00
52747 O. rugosa var. prostrata Pooyamkutty Ernakulam 0.47 ± 0.01
52754 O. rugosa var. prostrata Aluvamkudi Pathanamthitta 0.59 ± 0.01
52749 O. rugosa var. prostrata Brymore Thiruvananthapuram 0.24 ± 0.02
69917 O. rugosa var. prostrata Bonacaud Thiruvananthapuram 0.00 ± 0.00
52737 O. trichocarpon Rosemala Thiruvananthapuram 0.00 ± 0.00
69959 O. trichocarpon Plappally Pathanamthitta 0.32 ± 0.01
69965 O. trichocarpon Pampa Pathanamthitta 0.97 ± 0.02
52721 O. villosa Chemmunji Thiruvananthapuram 0.00 ± 0.00
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such as O. mungos, O. mungos var. angustifolia, O. eriantha, O. grandiflora, O. pectinata, O. trichocar-pon, and O. caudata (Renjith et al., 2013). This study is the first systematic CPT screening in O. barnesii, O. hirsutula, O. incarnata, O. radicans, and O. villosa. The four first-time screened Ophiorrhiza species (O. barnesii, O. incarnata, O. radicans, O. villosa) and two other species (O. caudata, O. grandiflora) showed zero or non-detectable levels of CPT (Table 1). Ophiorrhiza species with zero or near-zero levels of CPT can be ruled out as its primary sources.
The integrity of the analyte (CPT) (Figure 1) was confirmed by its isolation from the MeOH extract of O. mungos var. angustifolia and spectral characterization. A pale yellow powder, IR (λmax, cm−1): 3430, 1737, 1650, 1600, 1579, 1437, 1156, 1040; 1H NMR (δ): 5.28 (H-5), 8.66 (H-7), 8.10 (d, H-9, J = 8.0), 7.70 (t, H-10, J = 7.2), 7.85 (t, H-11, J = 7.6), 8.16 (d, H-12, J = 8.4), 7.37 (s, H-14), 5.41 (dd, H-17 J = 16.4, 23.4), 0.90 (t, H-18, J = 7.2), 1.89 (H-19), 6.30 (H-OH); 13C NMR (δ): 152.6 (C2), 145.5 (C3), 50.1 (C5), 129.7 (C6), 131.5 (C7), 128.0 (C8), 128.4 (C9), 127.5 (C10), 130.26 (C11), 129.0 (C12), 148.1 (C13), 96.7 (C14), 150.0 (C15), 119.1 (C16), 156.8 (C16a), 65.3 (C17), 7.7 (C18), 30.7 (C19), 72.4 (C20), 172.2 (C21); LC-EI-MS, m/z: 349.52 (M + H+). These spectral data are in good agreement with litera-ture reports (Krishnan, Dileepkumar, Nair, & Oommen, 2014; Rehman et al., 2009). CPT standard and isolated CPT showed exactly similar Rf values on HPTLC. On relative distribution analysis, root (403.64 μg/g, dr. wt.) of O. mungos var. angustifolia showed highest CPT content compared to its stem (216.35 μg/g, dr. wt.) and leaves (192.47 μg/g, dr. wt.) (Table 3). But in C. acuminata highest CPT content was found in its leaves (0.4–0.5%, dr. wt.) compared to its seeds and bark (López-Meyer et al., 1994).
3. Experimental
3.1. Plant materialsThirty-eight accessions of 11 Ophiorrhiza species viz., Ophiorrhiza barnesii C.E.C. Fisch. (1 accession), O. caudata C.E.C. Fisch (1), O. eriantha Wight (3), O. grandiflora Wight (1), O. hirsutula Wight ex Hook.f. (1), O. incarnata C.E.C. Fisch. (1), O. mungos L. (8), O. pectinata Arn. (6), O. radicans Gardn. (1), O. trichocarpon Blume (3), O. villosa Roxb. (1), and three varieties viz., O. mungos L. var. angustifolia
Table 2. Recovery dataAccession No. CPT + extract (μg) CPT added (μg) CPT in extract
(measured, μg)% Recovery
52753 (O. mungos) 0.001291 – 0.001291 –
52753 + CPT 1 0.001638 0.00035 0.001641 100.16
52753 + CPT 2 0.001703 0.00045 0.001741 99.79
52753 + CPT 3 0.001923 0.00065 0.001941 101.29
52755 (O. mungos) 0.002286 – 0.002286 –
52755 + CPT 1 0.002632 0.00035 0.002636 100.17
52755 + CPT 2 0.002762 0.00045 0.002736 99.07
52755 + CPT 3 0.002933 0.00065 0.002936 100.11
100.10 ± 0.72
Table 3. Distribution of camptothecin in different parts of O. mungos var. angustifoliaAccession No. Species Location District CPT ± SD (μg/g, dr. wt.)52734 whole plant O. mungos var. angustifolia Periya Wayanadu 373.19 ± 0.04
52734 leaf O. mungos var. angustifolia Periya Wayanadu 192.47 ± 0.07
52734 stem O. mungos var. angustifolia Periya Wayanadu 216.35 ± 0.61
52734 root O. mungos var. angustifolia Periya Wayanadu 403.64 ± 0.09
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(Thwaites) Hook. f. (4), O. rugosa Wall. var. decumbens Gardner ex Thwaites (1), O. rugosa Wall. var. prostrata (D. Don) Deb & Mondal (6) were collected (January–April 2013) from the southern Western Ghats region in India. Ophiorrhiza specimens were identified by Dr K. Satheesh Kumar, one of the authors, and voucher specimens were deposited at the Institute Herbarium (TBGT) (Table 1).
3.2. ExtractionShade dried, powdered plant materials (whole plants, 38 accessions, 10 g each) were Soxhlet ex-tracted (separately) using 200 ml MeOH for 6 h. In order to find the relative CPT distribution, O. mun-gos var. angustifolia root, stem and leaves (52734; 10 g each) were Soxhlet extracted (separately) with MeOH (200 ml, 6 h). After extraction, solvents were fully removed using a rotary evaporator (Buchi, Switzerland) and yields of extracts (w/w) were recorded.
3.3. HPTLC analysisQuantification of CPT in Ophiorrhiza (whole plant) extracts was carried out using an HPTLC (CAMAG, Switzerland) made up of Linomat V sample applicator, twin trough plate development chamber, TLC Scanner 3, and WinCATS Software 4.03 (Renjith et al., 2013). Briefly, Ophiorrhiza extracts dissolved in MeOH were filtered through nylon 0.45 μ membrane filters (PALL Gelman Laboratory, India). Filtered extracts of O. mungos, O. mungos var. angustifolia, O. mungos var. angustifolia (52734, root, stem and leaf extracts) (5 μl or 0.5 μg each), O. trichocarpon (10 μl or 10 μg, 20 μl or 200 μg) and other Ophiorrhiza species (20 μl or 200 μg each) were applied onto silica gel HPTLC plates (60F-254, E. Merck, Germany, 20 × 10 cm, 0.2 mm thickness) as 6 mm wide bands with the automatic Linomat V sample applicator fitted with a micro syringe in N2 flow (application rate—150 nl/s, space between two bands—11 mm, slit dimension—6 × 0.45 mm, scanning speed—20 mm/s). CPT standard (Sigma Aldrich, India) was also applied along with the Ophiorrhiza extracts. Plates were developed up to 80 mm in 20 ml of EtOAc:CHCl3:MeOH (5:4.5:0.5, v/v) mobile phase in the twin trough glass chamber, under saturated conditions (30 min). These plates were scanned densitometrically at 366 nm (tung-sten lamp) using TLC Scanner 3 and the data were analyzed using WinCATS Software 4.03 (Table 1).
3.4. Data analysis, validationHPTLC method was validated in terms of accuracy, precision, repeatability, and linearity (Fischedick, Glas, Hazekamp, & Verpoorte, 2009; Reich & Schibli, 2007; Renjith et al., 2013; Thomas et al., 2010). CPT contents (Table 1) in Ophiorrhiza extracts were determined by means of the calibration plot, y = (2 × 106 + 06) x + 67.12, R2 = 0.999, made of standard CPT (at 0.001 μg/μl in MeOH). Linearity of CPT calibration curve in the range 0.0001–0.004 μg was ensured. Specificity was tested by repeated application of standard CPT. Rf values of CPT were reproducible and found to be same as the values observed for the CPT peak in Ophiorrhiza extracts (0.42 ± 0.01, n = 20) (Figure 2). Integrity and purity of CPT peak were also tested by spectral analysis. Recovery studies were carried out by the addition of three varying concentrations of CPT to pre-analyzed Ophiorrhiza extracts (O. mungos 52753, 52755) and they were analyzed under similar conditions. Repeatability of sample application (instru-mental precision) was assessed by applying an Ophiorrhiza extract on the HPTLC plate, developed under similar conditions, the same spot was scanned six times, and the % coefficient of variation was acceptable (Table 4). Robustness of the method was checked by slightly altering the mobile phase composition and plate developing distance. Two different mobile phases viz., 20 ml each of EtOAc:CHCl3:MeOH (5.0:4.5:0.5, v/v) and EtOAc:CHCl3:MeOH (4.5:5.0:0.5, v/v), were used and
Table 4. Repeatability (precision) data
Note: 52725: O. rugosa var. prostrate; 52744: O. mungos; 69956: O. pectinata.
Accession No.
Area Average area
SD % RSD1 2 3 4 5 6
52725 400.5 408.2 405.2 412.8 416.3 401.8 407.46 6.21 1.52
52744 1,054.2 1,043.1 1,055.2 1,022.9 1,060.2 1,032.2 1,044.63 14.66 1.40
69956 2,130.2 2,169.1 2,192.3 2,145.5 2,122.8 2,132.5 2,148.73 26.84 1.24
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developing distances were varied to 75, 85 and 90 cm. No considerable effect on the data (Rf values of CPT) was found. % RSD (SD of CPT area/average CPT area) × 100 (for 20 accessions, n = 6) were determined. Limit of detection (LOD, average of 3.3 × SD of CPT area/slope of calibration curve for 25 accessions, n = 6) and limit of quantification (LOQ, average of 10 × SD of CPT area/slope of calibra-tion curve for 25 accessions, n = 6) were determined for CPT (Haridas et al., 2015; Renjith et al., 2013; Thomas et al., 2010). HPTLC analyses were carried out at laboratory conditions, 21 ± 0.5°C and 40% relative humidity (Barigo Humidity Meter, Germany).
3.5. Isolation, characterization of CPTCPT (Figure 1) was isolated from the MeOH extract of O. mungos var. angustifolia (whole plant) by repeated column chromatography using hexane:ethyl acetate and characterized using FT-IR (PerkinElmer, USA), 1H-, 13C-, 2D-NMR (Bruker, Germany), and LC-MS (Thermo Finnigan, USA).
4. ConclusionsHigh demand of CPT as a drug necessitates a supply demand equilibrium and prompts to search for its new natural sources. A total of 82 accessions (phase I 44, phase II 38) of 14 species and 3 varie-ties in genus Ophiorrhiza collected from the southern Western Ghats region in India were screened for CPT (Renjith et al., 2013). O. mungos and O. mungos var. angustifolia are the two best sources of CPT in genus Ophiorrhiza. In this study, four (O. barnesii, O. incarnata, O. radicans, O. villosa) of the five first-time screened species showed zero or non-detectable levels of CPT. These screening data can be utilized to choose elite eco-/chemotypes of Ophiorrhiza species/varieties for multiplication, and drug development.
AcknowledgmentsWe acknowledge Kerala Forest Department for permitting us to collect Ophiorrhiza specimens from protected forest areas. R.R. and S.C.V. are DAE-BRNS/MS University research fellows.
FundingThis work was supported by Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India [grant number 2010/35/4/BRNS/1278] dated 11/08/2010.
Author detailsRenjith Rajan1
E-mail: [email protected] Chirakkadamoolayil Varghese2
E-mail: [email protected] Kurup1
E-mail: [email protected] Gopalakrishnan3
E-mail: [email protected] Venkataraman4
E-mail: [email protected] Satheeshkumar2
E-mail: [email protected] Baby1
E-mails: [email protected], [email protected] Phytochemistry and Phytopharmacology Division,
Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India.
2 Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India.
3 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, Maharashtra, India.
4 Department of Chemistry, Sri Paramakalyani College, Manonmaniom Sundaranar University, Alwarkurichi, Tirunelveli 627 412, Tamil Nadu, India.
Citation informationCite this article as: HPTLC-based quantification of camptothecin in Ophiorrhiza species of the southern Western Ghats in India, Renjith Rajan, Sibi Chirakkadamoolayil Varghese, Rajani Kurup, Roja Gopalakrishnan, Ramaswamy Venkataraman, Krishnan Satheeshkumar & Sabulal Baby, Cogent Chemistry (2016), 2: 1275408.
Cover imageSource: Authors.
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