Review on Camptothecin Producing Medicinal Plant: Nothapodytes Nimmoniana

Nothapodytes nimmoniana is an endangered medicinal plant widely distributed throughout the Western Ghats of India. The plant contains camptothecin (CPT) which is renowned anticancer drugs. Though, CPT found in many plant species but maximum amount of CPT has been reported from N. nimmoniana. Due to very good source of CPT, this plant has been explored for its Phytochemical, Biotechnological and Pharmacological aspects. Looking to the huge global demand for CPT, overexploitation of N. nimmoniana, unplanned deforestation, and lowest production of CPT from intact plant, reduction of seed germination, high market cost and not have economically feasible process of production has optimistic us to investigate this plant in a systematic manner. The proposed article can be utilized for the establishment of extraction methods and analytical protocol for CPT. Also, Bioreactors production of CPT using high yielding cell line of N. nimmoniana. The pharmacological data will be applicable for discovery of new Drug and development lead to novel compounds which are safe and effective.

. Firstly, camptothecin has been obtained from in vivo grown barks and leaves, further in-vitro grown tissue culture of N. nimmoniana 5,6 . Then, it has been proceed from different parts of plant in various quantity with greatest yield occurs in roots then stem, leaves and fruits 7,8,9 . Camptothecin is useful for treatment of cancers.
Topotecan and Irinotecan are derivative of Camptothecin alkaloid that alternatives to cure of various kind of cancers. The marketing sales of these derivatives were estimated $1000 million annually and 1000-1500 tons of N. nimmoniana wood chips used to synthesis for about 1 ton of raw material of camptothecin 10,11 . The greater exporter of N. nimmoniana is wood chips for CPT to other countries for industrialization along with more than 1000 tons reported trade volume and twice unreported trade volume were reported. The Exporters were reported that twings of species sold at US$0.16 -$0.24/ kg and further sell out by pharmaceutical industries at US rate $15000/kg in the market of universe 10,12 . Due to increasing pharmaceutical marketplace demands of camptothecin and rapid use in cancer disease around the world, population of N. nimmoniana have been under exploitation pressure therefore required conserve this plant by following strategies of mass cultivatation 6,7 . Interest in plant secondary metabolites and its anticancer properties has need to prevalent study of other anticancer compounds from N. nimmoniana getting increase attention such as 9-methoxy camptothecin, Scopoletin, 10-hydroxycampthothecin, acetylcamptothecin, â-sitosterol, scopoletin, trigonelline and pumiloside 13,14 . Individual plant species showed naturally camptothecin compound variability crosswise a broad ecological range 7 Plant tissue culture approaches create alternative prospect for CPT production lacking harvesting of in their natural habitat populations. This approach of N. nimmoniana maintains populations of harvested plant and rapid propagation for conservation purpose 15,16 . The localization of N. nimmoniana species , chemically characterization and identification of individuals which produced high concentration of CPT used for material produced by clonal propagation and cell lines growth with high amount of CPT 9,17,18,19 . Further experimentally efforts were reported on culture media selection, culture media composition optimization or enrichment for enhancement in vitro response as well as CPT production [10,16,20] . This medicinal plant has widespread variety of pharmacologic activities against like cancer, HIV, bacterial infection, inflammatory, malaria, fungal infection, free radicals and anemia 21 . Mechanism of Action of Camptothecin 22 It has essential mechanism of action as a result of inhibition of DNA topoisomerase -I enzyme by following manner in figure 2 68 . Biosynthetic pathway of Camptothecin 23 C a m p t o t h e c i n ( i n d o l e a l k a l o i d ) synthesized by shikmate and MVA/DXP pathway. In Shikate pathway, firstly made chorismate which is synthesize anthranilate and trypthophan by synthase enzyme than tryptophan synthesize trpamine by decarboxylase enzyme. In MVA/ DXP pathway firstly made Gerniol which is synthesize 10-Hydroxygeranial by geranial 10hydroxylase and loganin synthesize by loganic acid o-methyl transferees than secologanin synthesize by secolganin synthase. Both of intermediate typtamine and secologanin alternatively synthesize strictosidine by synthase enzyme and camptothecin is synthesized following by 3-s pumiloside and 10-deoxypumiloside. The biosynthesis pathway of Camptothecin given in figure 3.

Plant Description
The plant Nothapodytes nimmoniana is a small tree and 3-8 meter tall. Branches are a little slanting, corky and scar leaves. Flowering season is during the month of July to August and ripened fruits are available during the month of November to December [16,24,25] 29 and structure of some major phyto-constituents given in figure 5.

Camptothecin (CPT) Extraction and Isolation
Soxhlet extraction was done for the CPT from stems of N. nimoniana by using various solvents such as methanol, chloroform, DMSO, n-haxane, toluene, petroleum ether, ethyl acetate and acetone. The results showed that the camptothecin was occurs in maximum concentration in In results, the camptothecin was found in maximum concentration in chloroform(0.98%) and methanol extracts (1.12%) whereas camptothecin was found in minimum concentration in DMSO extract (0.18%). Camptothecin was not found in petroleum ether, water and n-hexane extracts 30 Various extraction method such as soxhlet apparatus extraction method, microwave-assisted extraction (MAE), ultrasonic sound extraction method and stirring extraction method were performed for Camptothecin (CPT) and 9-methoxycamptothecin (9-MCPT) extraction from N. nimmonimana using ethanol (90%, v/v) and methanol (90%, v/v). The extraction yield high frequency of CPT and 9-MCPT using methanol comparatively ethanol. The result showed CPT was obtained in maximum percentage (2.67%, w/w) through MAE extraction method. Microwave extraction method involve for 3-5 min, while stirring extraction and ultrasonic extraction method involve for 30 min. and Soxhlet extraction method involve for 30, 120 and 30 min, correspondingly to maximum proportion of CPT and 9-MCPT 31 . The direct injection capillary electrophoresis technique employed for the marker compound camptothecin prepared by DMSO solvent from N. nimmoniana. In results, camptothecin were successfully established and was obtained camptothecin in linear range 5 to 400 µg/mL. The monitoring of camptothecin concentration successfully proved for the duration of the development of the therapeutic plant 32 .
The methanolic extracts of various kind of camptothecin producing plant such as Camptotheca acuminate, N. nimmoniana and Ophiorrhiza pumila employed using reversephase HPLC technique coupled with photodiode array detection and electrospray-ionization iontrap mass spectrometry. In results, the detection of camptothecin 0.146 % in C. acuminata and 0.048 % in N. nimmoniana 33 . The desorption electrospray ionization technique performed for the detection of 9-methoxycamptothecin (9-MCPT) and camptothecin (CPT) from of N. nimmoniana. The different parts were taken like leaves, stems and bark. In results, CPT precursor ion obtained fragmentation at m/z 349 showed a loss of 44 Da and producing the product ion at m/z 305 and the 9-MCPT precursor ion obtained fragmentation at m/z 379 showed a loss of 44 Da and producing the product ion at m/z 335. So, the desorption electrospray ionization is advanced technique for analysis of camptothecin and its derivative. This method suggested that the ion intensities and high amount of compound present in bark compared to the leaves and stems 34 . The hot extraction method proceeds for the CPT production from N. nimmoniana. The plant parts have been stem and twigs. The extraction followed by different solvents. In results, the extracts gave CPT with 0.15% yield 35 . The compound specific extraction of marker compounds by Accelerated Solvent Extractor (ASE). Two medicinal plants Nothapodytes nimmoniana and Piper nigrum was selected for the extraction of camptothecin (CPT) from stem and piperine from the fruits. The results showed the Effects of different temperatures improved yield high amount of CPT at 60-80p C compared to piperine yield at 37-40p C from this extraction method. The total CPT content was 0.1875g 100 g/dw. The retention time were obtained for CPT at 5.9326 ± 0.051 min. and for piperine at 7.7694 ± 0.0900 min 36 . The large scale isolation of camptothecin from Nothapodytes foetida: An improved process, through hot extraction process and precipitation process with some organic solvents. The camptothecin was yield upto 0.15% through the precipitation with a mixture of acetonitrile and dichloromethane 37 .

Phytochemical Analysis
The CPT accumulation, in particular organ, via variable extraction method and in vitro propagation method both qualitatively and quantitatively by TLC and HPLC analysis confirm the existence of CPT in dry leaf, fresh barks, induced callus and somatic embryo. The results showed that bark extracts possessed a maximum amount of CPT, i.e. 377.77 ìg/g. and soxhlet bark extract possesses the maximum amount of CPT 38 . The quantification of camptothecin was done by the liquid chromatography mass spectrometry (LCMS) from N. nimmoniana. In the results, the highest production of camptothecin was present in bark of stem (303.81ìg/ml) while in leaves (31.75 ìg/ml) 28 . The quantification of compounds 9-methoxycamptothecin and camptothecin (CPT) was obtained from N. nimmoniana by semi preparative HPLC. Camptothecin content was collected at 15 to 16 min and 9-methoxycamptothecin was collected at 20 to 25 min. The purification of the specific compounds was obtained by liquid chromatography-mass spectrometry (LC-MS) analysis technique. The purification was obtained 95% for CPT as compared to 9-MCPT 39 . The quantification technique HPLC analysis was performed for detection of the camptothecin content from the bark part of N. nimmoniana   9-Methoxy-camptothecin stem bark 3.
Pumiloside fruits , stem bark nimmoniana was contributed CPT (0.27% dw) and 9-MCPT (0.11% dw) rather than stem, root and leaves. In another plant parts like young seeds yield high amount of CPT (0.32% dw) and 9-MCPT (0.16% dw) compared to old seeds. The young seeds was contributed CPT (0.11% dw) and 9-MCPT (0.04% dw). The higher amount of CPT (0.42% dw) and 9-methoxycamptothecin (0.18% dw) were contributed in the young seeds 42 . The variation of camptothecin of the distribution in N. nimmoniana by means of gender, age and seasonality. In this report they initiate considerable variation in CPT substance along with individuals and chemically screening was done for identification of rich sources of the camptothecin. In results, the fairy good concentration of CPT from two year old seeds obtained in root tips (0.4%) compared to mature leaves (0.21%), immature leaves (0.20%), wood (0.20%) and root bark (0.16%) 9 [25] . The sophisticated technique high performance thin layer chromatography (HPTLC) for the quantification and validation of CPT. This validation was detection for precision, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy and repeatability. The peak was shown linear with the selection of 80-480 ng/spot and also shown correlation coefficient 0.998±0.020. The Repeatability was found 1.01 (% CV) of standard and 1.08(% CV) of sample. Limit of quantitation (LOQ) and limit of detection (LOD obtained 80 ng/spot and 40 ng/spot. The accuracy was determined through the typical proportion of recovery of 99.13% 44 . The quantification analysis technique for CPT in N. nimmoniana collected from various regions by high performance thin layer chromatographic technique. The following analysis was given the calibration curve for CPT that was linear in scale with the range of 0.3-3 ìg/ ml with the correlation coefficient of 0.982 45 .The studies of many medicinal plants like N. nimmoniana, Ophiorrhiza sp. was given for production of CPT. In results, N. nimmoniana was contributed overall camptothecin taken from sum of stem, leaves and roots was obtained 0.170% in dry weight and 9-methoxy camptothecin was obtained 0.002% in dry weight. The results suggested that camptothecin (CPT) and 9-methoxy camptothecin (9-ACPT) was accumulated in maximum percentage in N. nimmoniana 46   containing media obtained 31.3 g/l DW in shaking condition of flask after 20 days. The camptothecin and 9-methoxycamptothecin shown maximum amount 0.035 mg/ml and 0.026 mg/ml in the medium supplemented with NAA 50 . The accumulation of camptothecines and 9-methoxy camptothecine from callus cultures of from leaf explants of Nothapodytes foetida. The callus showed that the presence of CPT (2.893±2.39mg %) and 9-MCPT (0.4±0.4mg %). The result showed the quantity of camptothecine and 9-methoxy camptothecine produced in callus culture was low, than the intact plant 51 . The first time organogenesis generated on media supplemented with Thidiazuron (TDZ) revived from the callus cultures of Nothapodytes foetida. The dual role of Thidiazuron (TDZ) promoting proliferation of shoots and shoots differentiation from the callus. The enhanced production of CTP in regenerated plant of N. nimmoniana is using different media culture like semisolid and liquid cultures. The primary shoot proliferation obtained on phillps and Collins (L2)+ TDZ(0.44µM)+BAP(2.22µM)+Lglutamine(0.03mM) and respond 85.0% of shoots. The results showed that liquid culture are best for enhanced multiplication [18] . The multiplication of cell suspension culture for increased production of CPT from N. nimmoniana, culture optimized through different nitrogen content and different sugar feeding such as sucrose, fructose, glucose and maltose. In this study, MS medium optimized which was modified with supplementary 2 % sucrose (mix after 12 day) with 3% sucrose, a nitrogen source 50/10 mM NH4/NO3, 0.5 mM phosphate, 0.93 lM kinetin and 10.74 lM naphthaleneacetic acid (NAA). This modified culture medium produced higher camptothecin content about 1.7-and 2.3-fold more than following by the control, respectively. The result showed that the media enriched with high nitrate produced the biomass, while media with ammonium content enhanced CPT content and in case of sugar, sucrose supports maximum accumulation of CPT 20 . The callus was obtain in a variety of Murashige and Skoog (MS) media with various plant growth hormones such as BA, NAA, kinetin, Biotin and 2, 4-D. In results, the media supplemented with IAA and IBA were not obtained callus development. The most selectable callus culture was obtained 0.61% from immature leaves as compared to mature leaves 52 . N. nimmoniana also contributed in-vitro embryo culture and seed germination. The seed germination was achieved in highest percentage (69%) from uncoated seeds treat with concentration of 10mg/L gibberellic acid. The medium supplemented with Kn (1.0mg/L) and NAA (0.2mg/L) was found maximum frequency of seed germination 53.

The Endophtyic fungus for CPT production
The endophytic fungi strains were found about 52 kinds of strains isolated from mature and immature twigs of N. nimmonian collected from the regions of the Jammu and Mahabaleshwar The bioreactor studies was done for increased camptothecin content from Entrophospora infrequens, an the endophytic fungus and analyzed for accumulation of the CPT 54 . The endophytic fungus Neurospora sp. was studied isolated from seeds of N. nimmoniana cultivated in Sabouraud liquid media culture flask kept in rotatory shaker for shaking condition and estimated production of camptothecin. The confirmation of this compound from fungus culture was analyzed by chromatographic and spectroscopic analysis (LC-MS) and ion intensity of camptothecin showed m/z 349(M+H) 55. The endophytic fungi strains isolated from different plant parts given in table 5.

N. Nimmoniana: Pharmacological Studies Antimicrobial activity and Antiglycation activity
The antibacterial activity of stems and leaves from N. nimmoniana with different extraction solvents i.e. methanol, chloroform and petroleum ether. The zone of inhibition of stems showed in range 9-15 mm in methanol compared to other solvents. In results suggested that the stem methanol extracts were most efficient against microbial activity 57 . The Antimicrobial activity was shown in stem bark extract against Escherichia coli (MIC at 25mg/ml) and Pseudomonas aeruginosa (MIC at 25mg/ml) and in leaves against Pseudomonas aeruginosa (MIC at 50mg/ ml) by using Tetrazolium micro plate microbial viability assay. The stem bark extract and leaves extract was also shown the antiglycation activity by producing glycation end product. This end product was decreased due to the stem bark extract (IC50 at 10.356ìg/ml) and leaves extract (IC50 at10.995ìg/ ml) 28.

Antimalarial activity
The camptothecin has been effective and precise topoisomerase -I inhibitor drug against malaria parasites in vitro. In this study they were found principle action following by protein-DNA complexes trapped through camptothecin and repressed nucleic acid biosynthesis. Camptothecin was cytotoxic to P. falciparum, with an Ec 50 value of 32 µM and 36 µM 58.

Anti-inflammatory activity
The anti-inflammatory activity of the Nothapodytes nimmoniana by in vivo animal model following by carrageenan -was induced hind paw edema method in rats. The extracts were showed anti-inflammatory compared to control and standard ibuprofen induced group. These drugs were administered by oral rout. The result showed that ethanolic extract possess activity against inflammation which more efficient compared with petroleum ether extract and inflammation decreased significantly with the dose of ethanolic extract (200 mg/kg) compared to the standard ibuprofen 58.

Immunomodulatory activity
The endophytic fungus Entrophospora infrequens isolated from N. nimmoniana which possess immunomodulatory activity in vitro and in vivo (in Balb/c mice). This endophytes determined for production of camptothecin(CPT). In results, CPT-producing endophytic fungus was responsible for the immunomodulatory potential isolated from N. nimmoniana 60,69 .

Antioxidant activity
The methanol extracts of leaf and stem of Nothapodytes nimmoniana showed Antioxidant activity were determined using various in vitro models like DPPH (1,1-diphenyl-2-picryl hydrazine) ABTS•+ (2,2'-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) Ferrous iron chelating ability, Reducing power. The result showed significantly antioxidant activity was found in stem 84.35 % to 250 ìg/ml by DPPH method as compared to the standard and different parts of Nothapodytes nimmoniana 61 . The methanolic extracts of different parts of N. nimmoniana showed significant antioxidant activity. Antioxidant activity of methanolic extracts were determined using various in vitro models like Diphenylpicrylhydrazyl (DPPH) radical, nitric oxide radical, superoxide radical and peroxide radical scavenging activity. The reducing power, anti-lipid peroxidation potential and total phenolic content of methanolic extracts were also determined. Among the various plant parts phenolic content ranged from 281.0 to 454.0 mg gallic acid equivalent (GAE) /100 g dry sample with maximum content found in methanolic extract of fruits (454.0 mg GAE/gm DW). Fruits shown maximum antioxidant activity with an IC50 value of 0.177 ± 0.2 mg/mL for DPPH radical, 0.177 mg/mL for H 2 O 2 radical, 0.167 mg/ mL for superoxide radical and 0.175 mg/mL for nitric oxide radical. Fruits showed maximum anti-lipid peroxidation effect (0.362 mg/mL) with higher reducing potential 3.65. The results were significant as compared to standard antioxidants such as l-ascorbic acid and á-tocopherol 62 .

Anticancer/cytotoxic activity
Nothapodytes nimmoniana produced the alkaloid compound camptothecin(CPT). This study suggested that CPT inhibited specifically DNA topoisomerase enzyme and caused DNA damage. This drug effectively demoralized various types of cancer disease 63 . The principle action of camptothecin(CPT) produced by Nothapodytes nimmoniana which activated S or G2-M arrest and the homologous recombination repair pathway in tumor cells 64 . Nothapodytes nimmoniana produced new compound, 10-methoxy-9-nitrocamptothecin which showed anticancer activity. In results, this compound showed great anticancer activity along with enhancing the cellular accumulation of DNA damage 65 . The 18 known compounds in which acetylcamptothecin was a new naturally occurring alkaloid compound from Nothapodytes foetida (Wight) Sleumer. Among these camptothecin, scopoletin, O-acetylcamptothecin and 9-Omethoxy camptothecin showed significant anticancer activity 42. The endophytic fungus accumulated such compounds which showed anticancer activity isolated from Nothapodytes nimmoniana. The results showed effectively influenced against all cancer cell lines 26,66 . The anticancer effect of its hydro alcoholic extract on HeLa cell lines, the results showed that the cytotoxic effect on HeLa cell lines was determined using MTT assay, where IC50 for leaves, stem bark and standard CPT was 178ìg/ìl, 1540ìg/ìl, and 29ìg/ìl respectively 28.

Nephro Protective and Hepatoprotective activity
The evaluation of new activities was determined nephro-protective and hepatoprotective activities of Nothapodytes nimmoniana. The phytoconstituents like alkaloid (camptothecin) and flavonoids shown nephroprotective and hepatoprotective activity from N. nimmoniana 67 .

CoNCluSIoN
Nothapodytes nimmoniana is a natural source of Camptothecin(CPT). Looking to its high demands, this plant needs exploration of the phytoconstituents aspects and therapeutic potential. The exploration of biotechnological properties of N. nimmoniana through cell suspension culture and callus culture are used as alternative for camptothecin production which is one of the medically important alkaloid and is of great demand in the pharmaceutical world as it is used for the formation of anti-cancer drugs. Therefore, this plant species needs improving their conservation using various techniques like maintaining genetic impact of species and micro propagation technique. The present review study revealed that Nothapodytes nimmoniana is alternative sources of natural drug for treatment of cancers.

Future Prospects
Vaious phytochemical extaction methods need to optimize comparatively for production of Camptothecin. Plant biotechnological techniques like cell suspension and callus culture are occupying better results for the production of Camptothecin and another phytoconstituents like 9-methoxy Camptothecin, 9-amino Camptothecin,10-hydroxyl Camptothecin, â-sitosterol, trigonelline, scopoletin and pumiloside. Advance interest of development of callus cultures by using up scaling bioreactor and hairy root cultures produce high amount of alkaloid compounds. Endophytes also have industrial applications for the production of high yield of CPT. Optimization of cultures through modified elicitors and precursor feeding enhance production of CPT and other alkaloid compounds that will be great interest of exploration. Micro-propagation of N. nimmoniana is need for conservation of plant species that reduce overexploitation.