CATHARANTHUS ROSEUS AND PROSPECTS OF ITS ENDOPHYTES: A NEW AVENUE FOR PRODUCTION OF BIOACTIVE METABOLITES

CATHARANTHUS ROSEUS AND PROSPECTS OF ITS ENDOPHYTES: A NEW AVENUE FOR PRODUCTION OF BIOACTIVE METABOLITES

Meenakshi Koul, Neha S. Lakra, Ramesh Chandra and Sheela Chandra*

Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi- 835 215, Jharkhand, India

ABSTRACT: Catharanthus roseus is a medicinal herb found in many tropical and subtropical regions around the world. This plant produces a diverse array of secondary metabolites that are pharmaceutically important like vinblastine and vincristine used as chemotherapeutic agents in the treatment of several types of cancers. Low yield of these vinca alkaloids from the plant in vivo and the challenges to meet their high demand worldwide led researchers to develop various in vitro techniques like hairy root culture, callus cultures, shoot cultures, metabolic engineering and regulation studies to increase their production. The present review gives an account of the various phytochemicals derived from the plant and the pharmacological aspects of secondary metabolites studied. Present review also highlights the biotechnological prospects of an efficient and alternative means of production of valuable metabolites from Catharanthus roseus and also from rich microflora residing inside the plant tissues. It gives an emphasis on the need of exploration of diverse niches of endophytes.

Vinca alkaloids, Endophytes, Anti-tumor activity, Antidiabetic activity, Hypotensive activity

INTRODUCTION:Catharanthus roseus is a medicinal plant belongs to the family Apocynaceae native and endemic to Madagascar. The plant is also known by the names such as Vinca rosea, Ammocallis rosea and Lochnera rosea. The plant has been put to traditional use for the treatment of a wide variety of ailments worldwide since ages ¹. The plant bears active phytoconstituents and exhibits various pharmacological activities like antidiabetic, antioxidant, anti-hypertensive, antimicrobial, cytotoxic etc. Catharanthus roseus produces a spectrum of terpenoid indole alkaloids (TIAs) vinblastine and vincristine, the anticancer lead molecules.

Being a source of these important secondary metabolites, an extensive study has been carried out on C. roseus. The present review provides a description of the secondary metabolites derived from this plant, its pharmacological activities, the biotechnological approaches undertaken to enhance the production of TIAs and the prospects of potential endophytes residing inside the host tissue.

Active Constituents:

Alkaloids: C. roseus is known to be a source of about 150 active alkaloids out of which vincristine, vinblastine and vindiscline are of prime importance because of their use in the treatment of Cancer ². Vinca alkaloids vincristine and vinblastine are used in chemotherapy with vincristine being used for acute lymphocytic leukemia, both Hodgkin and non-Hodgkin lymphomas and vinblastine being used as the major component in chemotherapy for germ cell, breast, bladder and some types of brain malignancies³.

Vinblastine and Vincristine shown in (Figure 1) are anti-tubulin drugs that act by suppressing the spindle microtubule dynamicity in the cells during mitosis thereby arresting cell division and causing cell death ⁴.

VINCRISTINE                                                    VINBLASTINE

FIGURE 1: ANTI LEUKEMIC ALKALOIDS ISOLATED FROM C. ROSEUS

Vinblastine and vincristine have been isolated in a pure form from C. roseus L. Don by the use of several chromatographic techniques like vacuum liquid chromatographic column on silica gel: aluminium oxide (1:1) mixed bed vacuum liquid chromatography (VLC), charcoal column, and finally purified by centrifugally accelerated radial chromatography (Chromatotrone) ⁵. Several other methods like high performance liquid chromatography ^{6, 7} and supercritical fluid extraction⁸ have also been devised to efficiently quantify these alkaloids in the plant.

Vinblastine and vincristine are the dimers formed by the coupling of Monoindole alkaloids such as catharanthine and vindoline found abundantly inthe aerial parts of the plant ⁹. The biosynthetic pathway of these alkaloids as in (Figure 2) has been found to be under strict developmental regulation in the plant¹⁰. Various studies on the regulation of the biosynthetic pathways as summarised in Table 1 revealed that terpenoid- indole alkaloid biosynthesis is subjected to different enzymatic and genetic regulation in the plant system.

FIGURE 2: TERPENOID INDOLE ALKALOID BIOSYNTHESIS IN PLANT SYSTEM

The enzymes are abbreviated as follows: TDC – Tryptophan decarboxylase, STR – Strictosidine synthase, SGD – Strictosidine glucosidase, T16H– Tabersonine 16-hydroxylase, NMT - 16-methoxy-2,3-dihydro-3-hydroxy tabersonine N-methyl transferase, D4H - Vindoline-4-hydroxylase, DAT- Deacetylvindoline O-acetyltransferase

TABLE 1: STUDIES ON ENZYMES INVOLVED IN TIAS BIOSYNTHESIS IN CATHARANTHUS ROSEUS

Further, subcellular localization of these enzymes is also implicated in the regulation of the TIA biosynthetic pathway. Tryptophan decarboxylase and strictosidine synthase involved in the synthesis of strictosidine are both found in the cytosol.

16-methoxy-2, 3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the vindoline biosynthesis are localized in the chloroplasts of leaves.

Acetyl-coenzyme-A-deacetylvindoline-O-acetyl transferase catalysing the last step in vindoline biosynthesis is also a cytoplasmic enzyme ²⁰. Most recently it has been reported that the entire production of catharanthine and vindoline occurs inside young leaves where the former accumulates in the leaf wax exudates and the latter within the leaf cells thus making leaves of this plant an important source of chemotherapeutic drugs ²¹.

The chemical synthesis involving direct coupling of vindoline and catharanthine and asymmetric total synthesis of vindoline and vindorosine based on a unique intramolecular [4+2]/[3+2] cycloaddition cascade of 1,3,4-oxadiazoles have also been studied as a means for the production of terpenoid indole alkaloids like vinblastine, vincristine ^{22, 23}.

Polyphenolics: Study of non-coloured phenolics from seeds, stems, leaves and petals of C. roseus and evaluation of their antioxidant activity led to the characterization of three caffeoylquinic acids and some flavonol glycosides with structures as in Figure 3. The scavenging ability of different plant matrices was assessed and a concentration-dependent protective effect was observed for seeds and tissues, with petals found to be most active followed by seeds and leaves, indicating their potential for use in food, pharmaceutical and cosmetic industries ²⁴.

FIGURE 3: CHEMICAL STRUCTURES OF THE PHENOLIC COMPOUNDS IDENTIFIED IN C. ROSEUS

Anthocyanins: The production of anthocyanins has been described in vivo as well as in vitro from C. roseus. The major anthocyanins have been identified as the 3-O-glucosides, and the 3-O-(6-O-p-coumaroyl) glucosides of hirsutidin, malvidin and petunidin shown in Figure 4, respectively both in vivo and in vitro plant cell cultures ²⁵. Besides these the presence of tricin, a flavone, was reported in the mature C. roseus petals ²⁶.

FIGURE 4: ANTHOCYANINS FOUND IN C. ROSEUS

Steroids: Studies have shown that crown gall cells of octopine and nopaline-types derived from C. roseus (L.) G. Don produce brassinosteroids with the main components identified as brassinolide and catasterone. Brassinosteroids the steroidal growth-promoting plant hormones have been found to be useful in agriculture for increasing crop production, and improving stress resistance of crops against drought, chilling and pesticides ²⁷.

Flavonoid glucosides: Four new flavonoid glucosides, 3’,4’-di-O-methylquercetin-7-O-[(4”→13”’)-2”’,6”’,10”’,14”’-tetramethylhexadec-13”’-ol-14”’enyl]-β-D-glucopyranoside, 4’-O-methyl kaempferol-3-O-[(4’’→13”’)-2”’,6”’,10”’,14”’-tetramethylhexadecane-13”’-olyl]-β-D-gluco pyranoside,3’,4’-di-O-methylbutin-7-O-[(6”→1”’)-3”’,11”’,-dimethyl-7”’-methylenedodeca 3”’, 10”’ dienyl]-β-D-glucopyranoside and 4’-O-methylbutin-7-O-[(6”→1”’)-3”’,11”’,-dimethyl-7”’-hydroxymethylenedodecanyl]-β-D-glucopyranoside were isolated from the methanol extract of C. roseus hairy roots for the first time. These new flavonoids were shown to inhibit MMP-9 activity and TNF-α production in THP-1 cells implying to their use as potential anti-inflammatory medication ²⁸.

Iridoid glucosides: C. roseus is known to accumulate monoterpene indole alkaloids that are derived from the coupling of tryptamine and iridoids like loganin and secologanin (Figure 5) ²⁹. The distribution of these iridoids at sub-cellular levels was studied in secologanin accumulating C. roseus cells and secologanin was found to be stored exclusively in the vacuoles ³⁰.

LOGANIN

SECOLOGANIN

FIGURE 5: IRIDOID GLUCOSIDES FOUND IN C. ROSEUS

Novel metabolites from cell cultures of C. roseus: Catharanthus roseus is not only an important source of the metabolites discussed above but has also been studied as the source of some novel active metabolites.

Some of the novel metabolites obtained from the cell cultures of C. roseus and the approaches used to produce them are discussed in Table 2.

TABLE 2: NOVEL METABOLITES FROM C. ROSEUS CELL CULTURE

 Pharmacological Studies:

Antidiabetic activity: Study of a dichlormethane: methanol extract (1:1) of leaves and twigs of C. roseus in Streptozotocin-induced diabetic rat models exhibited hypoglycemic activity with the improvement in decreased enzymic activities in liver of diabetic animals. Increased levels of lipid peroxidation caused during oxidative stress were also normalized by extract treatment ³⁴.

The leaf juice of C. roseus showed a dose-dependent reduction in blood glucose in both normal and diabetic rabbits when compared to the standard drug, glibenclamide. The mechanism of action was probably due to enhanced secretion of insulin from the ß-cells ³⁵.

Study of C. roseus, Azadirachta indica and Allium sativum showed significant antidiabetic activity for all three medicinal plants compared with the patent drug glimepride supporting their usage as herbal medicines for the diabetes by Ayurvedic physicians³⁶. An investigation to study the impact of C. roseus leaves on the management of diabetes mellitus in about 20 type-2 diabetics residing in Dharwad city revealed a significant reduction in the fasting blood glucose, post prandial blood glucose, total cholesterol, low density lipoprotein cholesterol and triglyceride levels of the diabetic subjects ³⁷.

A study carried out to investigate the antidiabetic and hyperlipidemic potential of C. roseus on alloxan induced diabetes in male albino rats revealed the therapeutic value of the aqueous flower extracts of C. roseus to combat the diabetic condition in rats ³⁸.

Anti-tumor activity: A U.S. government screening program incidentally discovered C. roseus alkaloids vinblastine and vincristine, as well as some synthetic analogs as highly toxic chemotherapy drugs ³⁹. Semi synthetic analogs, vinorelbine (VRLB) and vindesine (VDS), obtained from the active compounds showed potential activity against leukemias, lymphomas, advanced testicular cancer, breast cancer, lung cancer and Kaposi’s sarcoma in combination with other chemotherapeutic drugs ⁴⁰.

Vinflunine, a bifluorinated derivative of vinorelbine exhibits a superior anti-tumor activity compared to other vinca alkaloids viz. vinorelbine and vinblastine. The mechanism of mitotic block is unknown but it is hypothesised that the anti-tumor activity is due to the decrease in microtubule dynamicity during mitosis and the increase in time centromere spends in the resting state during cell cycle. This novel vinca alkaloid is currently under Phase II clinical trials ⁴¹.

 Cytotoxic activity: C. roseus was investigated for its cytotoxic activity by using MTT assay against Human Colorectal Carcinoma cell line (HCT 116). The study showed dose dependent cytotoxic activity of the methanol extract of C. roseus leaves with the chloroform extract showing the highest activity ⁴². The aqueous extract of C. roseus leaves standardised to Vinblastine was found to inhibit the proliferation of Jurkat cell line indicating the efficacy of the extract for modulating normal and transformed immune cells in leukemia patients ⁴³. Three new dimeric indole alkaloids isolated from the whole plants of C. roseus have been evaluated for their cytotoxic activities against human breast cancer cell line MDA-MB-231 ⁴⁴.

Antimicrobial activity: Ethanol extract of the C. roseus flowers was studied for its wound healing potential in Sprague Dawley rats and the study showed that the extract had properties rendering it capable of promoting accelerated wound healing activity. Besides this the increased wound contraction and tensile strength, increased hydroxyproline content and antimicrobial activity further supported the topical use of C. roseus in wound treatment and management ⁴⁵.

Crude extracts from different parts viz leaves; stem, root and flowers of C. roseus were tested for antibacterial activity. The leaf extract showed significantly higher activity suggesting that bioactive compounds of C. roseus can be a potentially exploited as antibacterial agents. Gram (-) strains were found to be more sensitive than the Gram (+) bacteria ⁴⁶.

A study conducted to determine the antibacterial activity of crude extracts from different parts (leaves, stem, root and flower) of C. roseus against several bacteria of clinical significance indicated that the extracts prepared from the leaves showed better efficacy, the ethanolic extracts were more active against almost all the test microbes and Gram-positive bacteria were found to be more sensitive than the Gram-negative ones ⁴⁷.

The plant parts, leaves, stems, roots and flowers of two varieties of Catharanthus roseus (L.) G. Don. "rosea" and "alba" were tested for their antibiogram by using different solvents (methanol, acetone and ethyl acetate). The variety “rosea” was found to have a better antibiogram than the alba variety.

Of the three solvents ethyl acetate extracts of different plant parts were found to have best antibiogram followed by methanol and acetone extracts ⁴⁸.

In a study aimed to investigate some of the anti-microbial properties of C. roseus against microorganisms like Pseudomonas aeruginosa NCIM 2036, Salmonella typhimurium NCIM 2501, Staphylococcus aureus NCIM 5021 it was found that the extracts from the leaves of this plant can be used as prophylactic agent in many of the diseases, which sometime are of the magnitude of an epidemic ⁴⁹. The leaf extract of C. roseus has also been shown to have significant fungitoxic activity against Macrophomina phaseolina and Sclerotium rolfsii the causative agents of the root rot disease in chickpea (Cicer arietium L.). The extracts strongly inhibited the mycelial growth in both the fungi at 50, 75 and 100% concentration when compared with the control⁵⁰.

In a study to explore the antiplasmodial potential of C. roseus L, Coccinea grandis, Thevetia peruviana, Prosopis juliflora, Acacia nilotica, Azadirachta indica (Abr. Juss) and Morinda pubescens, the bark extract of A. indica (Abr. Juss) was found to have excellent antiplasmodial activity followed by leaf extract of A. indica (Abr. Juss) and leaf extract of C. roseus L ⁵¹.

Antioxidant potential: The effects of triadmefon treatment, a triazole compound on the antioxidant potentials and root alkaloid ajmalicine content were studied in two varieties of C. roseus, rosea and alba. The treatment with triadimefon increased the antioxidant potential as well as the indole alkaloid ajmalicine (more in the rosea variety than the alba variety) content. Results suggested that triadimefon may be a useful tool for increasing alkaloid production in medicinal plants ⁵².

A study to comparatively evaluate the antioxidant potential of ethanolic extracts of the roots of the two varieties of C. roseus and C. alba using different systems of assay, e.g. Hydroxyl radical-scavenging activity, superoxide radical-scavenging activity, DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical- scavenging activity and nitric oxide radical inhibition method was performed. The results revealed that the root extracts prepared in ethanol exhibited satisfactory scavenging effect in all the radical scavenging assays in a concentration dependent manner and C. roseus showed more antioxidant activity than Catharanthus alba ^53.

Hypotensive activity: The leaf extracts of C. roseus were investigated for the hypotensive and hypolipidemic activity in adrenaline induced hypertensive rats in a study. C. roseus leaf extract was found to have significant effect on each cardiovascular parameter after investigation with regard to hypotensive and hypolipidemic effect ⁵⁴. The dry leaf powder of C. roseus was investigated for its antihyperlipidemic and antioxidant efficacy in male albino Wistar rats. The results of the study suggested that C. roseus possesses a significant antihyperlipidemic and antioxidant efficacy by attenuating the biochemical and physiological alterations in Streptozotocin induced diabetic rats ⁵⁵.

Anthelmintic activity: Study of leaves extract of C. roseus showed potent antihelmintic activity in experimental adult earthworm Pheretima posthuma with the decrease in death time as the concentration increased. In the study, the control drug Piperazine citrate showed more potent anthelmintic activity compared to the methanol, aqueous, ethanol and ethylacetate extract ⁵⁶. The anthelmintic property of C. roseus was evaluated using Pheretima posthuma as an experimental model and Piperazine citrate as the standard reference.

Among the various test concentrations, ethanol extract 250 mg/ml showed significant anthelmintic activity with death time of 46.33 min as compared to the standard drug at 50 mg/ml that showed paralysis at 31.33 min and death time as 40.67 min. The investigation thus revealed that ethanol extract of C. roseus showed significant antihelmintic activity against Pheretima posthuma supporting the ethnomedical claims of C. roseus as an antihelmintic plant ⁵⁷.

Neuroprotective activity: A study was done to investigate the possible neuroprotective effect of C. roseus leaf extract against streptozotocin induced hyperglycaemia in the rat brain demonstrated that C. roseus leaf extract is an effective neuroprotective agent against diabetic oxidative damage as treatment with C. roseus reduced MDA, XO and Sorbitol DH production and increased glutathione levels significantly when compared to the streptozotocin induced diabetic-untreated rats ⁵⁸.

Antifertility efficacy: Oral administration of C. roseus Linn, leaf extract leading to widespread testicular necrosis, hyalinization of tubules and sertoli cell-only-Syndrome, notable reduction in glycogen and fructose levels in reproductive tissues confirmed the antifertility properties of C. roseus extract ⁵⁹. The petroleum ether extract of C. roseus leaves inhibited the estrogen induced gain in the uterine weight when administered along with estradiol into the female albino mice thus proving to be highly effective in suppressing pregnancy ⁶⁰.

Biotechnological approaches to enhance in vitro production of terpenoid indole alkaloids from C. roseus: Catharanthus roseus has been of much interest among the scientific and medical communities because of its pharmacological potential. The interest can be traced back to the mid-1950, when researchers began to study the plant for its reported antidiabetic properties. The pharmacological potency of the plant as discussed earlier is due to its varied active constituents and the plant being a sole commercial source of anti-cancer alkaloids vincristine and vinblastine ⁶¹ has made it an important subject of research.

Most of the scientific research done on C. roseus in the past years had been focussed towards the isolation and characterisation of TIAs, testing their bioactivity and study of their biosynthetic pathways. Low yield of these vinca alkaloids from the plant in vivo (0.0005%) ⁶² and the challenge to meet their high demand worldwide (3kg/annum) has led to the development of various in vitro techniques like hairy root culture, callus cultures, shoot cultures for their increased production. Besides these classical tissue culture techniques, metabolic engineering aspects have also been studied to improve the production of terpenoid indole alkaloids ⁶³.

Metabolic and biochemical engineering have been studied as a perspective for the creation of new cell lines producing TIAs in large scale bioreactors combined with efficient upstream and downstream processing ⁶⁴. Recently, a study to investigate the effect of various elicitors of hydroxylase, peroxidase, acetyltransferase and inhibitors of oxygenase on regulation of vinblastine biosynthesis in cell suspension cultures of C. roseus showed Hydrogen peroxide, Acetyl CoA, Benzotriazole to be very effective in enhancing the production of vinblastine⁶⁵.

Table 3 summarises some of the biotechnological approaches used to increase the terpenoid indole alkaloid production.

TABLE 3: BIOTECHNOLOGICAL APPROACHES TO INCREASE TIAS PRODUCTION

Endophytes:

A new avenue for production of bioactive metabolites: Despite these continuous efforts, the desired level of production of these metabolites has still not been achieved at optimum level, thereby making it necessary to bioprospect their new sources. Taking into consideration the limitations associated with the production of these metabolites in vivo and the need to preserve the world’s ever-diminishing biodiversity, a microbial source of a valued product may be easier, sustainable and more economical for the production of valuable metabolites, effectively reducing their market price. Endophytes, the microbes that colonize the internal tissues of the plants without causing any overt negative effects, could thus be the potential sources of these valuable bioactive metabolites ⁸⁰. The endophytes are a diverse group of microbes owing to the fact that almost all vascular plants examined to date have been found to colonize these microbes ^{81, 82}. Due to varied ecological niches of C. roseus, there is also a probability of diversity in endophytic localisation inside the plant tissues. The endophytes are sources of bioactive compounds like alkaloids, terpenoids, flavonoids, steroids etc that have importance in medicine, agriculture and industries as well ^{83, 84}. Table 4 gives an account of the bioactive compounds obtained from the endophytes islolated from Catharanthus roseus till date.

TABLE 4: BIOACTIVE COMPOUNDS OBTAINED FROM ENDOPHYTES ISOLATED FROM C. ROSEUS

Endophytes isolated from C. roseus not only yield these valuable therapeutic molecules but have recently been studied to improve the in planta content of terpenoid indole alkaloids like serpentine, ajmalacine, vindoline and vinblastine. The study showed that the bacterial endophytes isolated from this plant identified as Staphylococcus sciuri and Micrococcus sp.could possibly be used as bio-inoculants to increase the plant biomass and the content of key terpenoid indole alkaloids within the plant thus providing an efficient and economic means to overcome the gap between high demand and low supply of these vinca alkaloids globally ⁹¹.

Conclusion: Catharanthus roseus is an important medicinal plant with a wide range of uses. The dried plant extracts contain many alkaloids of medicinal use. These alkaloids are produced in very small quantities inside the plant although attempts have been made over the past years to increase their production through various biotechnological applications. The plant has been proven useful not only in the field of medicine but has also been recently put into use for the phytoremediation of radiocesium ¹³⁷Cs from low level nuclear waste ⁹².

The leaves of this plant have been found to be of immense medicinal use as most of the pharmacological activities of this plant are attributed to its leaves. So the cultivation as well as the conservation of this plant must be promoted on a large scale. Besides this alternative means, that are less time consuming, sustainable and more economical, must be developed and adopted for the production of these active constituents.

In the present times, when the emphasis is being placed on the use of natural materials in the control and treatment of various diseases and infections because of the undesirable side effects of synthetic drugs there is a need for further research especially on bioactive compounds, their production from alternative sources, methods for increasing their production, herbal remedies,  effectiveness of plants for various uses and bioprospecting new sources of natural bioactive products which can provide unlimited scope for the development of new drug leads. Endophytes could thus be exploited as the sources of the valuable secondary metabolites of medicinal, agricultural and industrial importance.

Owing to the huge microbial biodiversity of endophytes, these are still the less investigated group of microorganisms that need to be explored for their huge potential of being used as the sources of pharmacologically active therapeutic lead compounds.

References:

1.

1.            Aslam J, Khan SH, Siddiqui ZH, Fatima Z, Maqsood M, Bhat MA, Nasim SA, Ilah A, Ahmed IZ, Khan SA, Mujib A and Sharma MP: Catharanthus roseus (L.)G. Don. an important drug: its applications and production. Pharma Globale 2010;1(4): 1-16.

2.            Negi RS: Fast in-vitro callus induction in Catharanthus roseus- a medicinally important plant used in cancer therapy. Res J Pharma Bio Chem Sci 2011; 2(4): 597-603.

3.            Mann J: Natural products in cancer chemotherapy: past, presesnt and future. Nat Reviews 2002; 2: 143-148.

4.            Jordan MA, Thrower D and Wilson L: Mechanism of inhibition of cell prolifertaion by vinca alkaloids. Cancer Res 1991; 51: 2212-2222.

5.            Shams KA, Nazif NM, Azim NA, Shafeek KA, El-Missiry M, Ismail SI and Seif El Nasr M: Isolation and characterization of antineoplastic alkaloids from Catharanthus roseus L. Don. cultivated in Egypt. Afr J Trad CAM 2009; 6(2): 118-122.

6.            Hisiger S and Jolicoeur M: Analysis of Catharanthus roseus alkaloids by HPLC. Phytochem Rev 2007; 6:207-234.

7.            Siddiqui MJ, Ismail Z and Saidan NH: Simultaneous determination of secondary metabolites from Vinca rosea plant extractives by reverse phase high performance liquid chromatography. Pharmacogn Mag 2011; 7(26): 92-96.

8.            Sang KM, Park SW, Hong WH and Lee H: Isolation of vindoline from Catharanthus roseus by supercritical fluid extraction. Biotechnol Prog 1992; 8(6): 583-586.

9.            Renault JH, Nuzillard JM, Crouerour GL, Thepenier P, Hanrot MZ and Olivier LM: Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode. J Chromatography 1999; 849: 421-431.

10.            Chandra S and Chandra R: Engineering secondary metabolite production in hairy roots. Phytochem Rev 2011; 10: 371–395.

11.            Endo T, Goodbody A, Vukovic J and Misawa M: Enzymes from Catharanthus roseus cell suspension cultures that couple vindoline and catharanthine to form 3’,4’-anhydrovinblastine. Phytochem 1988; 27(7): 2147-2149.

12.            Luca VD, Fernandez JA, Campbell D and Kurz WG: Developmental regulation of enzymes of indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 1988; 86: 447-450.

13.            Carolis ED, Chan F, Balsevich J and Luca VD: Isolation and characterization of a 2-Oxoglutarate dependent Dioxygenase involved in the Second-to-last step in Vindoline biosynthesis. Plant Physiol 1990; 94: 1323-1329.

14.            Aerts RJ and Luca VD: Phytochrome is involved in the light-regulation of Vindoline biosynthesis in Catharanthus. Plant Physiol 1992; 100: 1029-1032.

15.            Waal AD, Meijer AH and Verpoorte R: Strictosidine synthase from Catharnthus roseus: purification and characterization of multiple forms. Biochem J 1995; 306: 571-580.

16.            Rischer H, Oresic M, Laakso TS, Katajamaa M, Lammertyn F, Diaz WA, Montagu MV, Inze D, Caldentey KO and Goossens A: Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. PNAS 2006; 103: 5614-5619.

17.            Bernhardt P, McCoy E and O’Connor SE: Rapid identification of enzyme variants for reengineered alkaloid biosynthesis in Periwinkle. Chem Biol 2007; 14(8): 888-897.

18.            Sottomayor M, Duarte P, Figueiredo R and Barcelo AR: A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus. Plant Sig Behavior 2008; 3: 899-901.

19.            Liscombe DK, Usera AR and O’Connor SE: Homolog of tocopherol C methlytransferases catalyzes N methylation in anticancer alkaloid biosynthesis. PNAS 2010; 107: 18793-18798.

20.            Luca VD and Cutler AJ: Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 1987; 85: 1099-1102.

21.            Roepke J, Salim V, Wu M, Thamm AM, Murata J, Ploss K, Boland W and Luca VD: Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. PNAS 2010; 107: 15287-15292.

22.            Ishikawa H, Colby DA, Seto S, Va P, Tam A, Kakei H, Rayl T, Hwang I and Boger DL: Total synthesis of vinblastine, vincristine, related natural products, and key structural analogues. J Am Chem Soc 2009; 131: 4904–4916.

23.            Sasaki Y, Kato D and Boger DL: Asymmetric total synthesis of Vindorosine, Vindoline and key Vinblastine analogues. J Am Chem Soc 2010; 132(38): 13533-13544.

24.            Ferreres F, Pereira DM, Valentao P, Andrade PB, Seabra RM and Sottomayo M: New phenolic compounds and antioxidant potential of Catharanthus roseus. J Agric Food Chem 2008; 56: 9967-9974.

25.            Piovan A and Filippini R: Anthocyanins in Catharanthus roseus in vivo and in vitro: a review. Phytochem Rev 2007; 6: 235-242.

26.            Vimala Y and Jain R: A new flavone in mature Catharanthus roseus petals. Indian J Plant Physiol 2001; 6(2): 187-189.

27.            Sakurai A and Fujioka S: Studies on biosynthesis of Brassinosteroids. Biosci Biotech Biochem 1997; 61(5): 757-762.

28.            Chung IM, Ahmad A, Ali M, Lee O, Kim MY, Kim JH, Yoon D, Peebles CA and San KY: Flavonoid glucosides from the hairy roots of Catharanthus roseus. J Nat Prod 2009; 72(4): 613-620.

29.            Oudin A, Courtois M, Rideau M and Clastre M: The iridoid pathway in Catharanthus roseus alkaloid biosynthesis. Phytochem Rev 2007; 6: 259-276.

30.            Contin A, Heijden RV and Verpoorte R: Accumulation of loganin and secologanin in vacuoles from suspension cultured Catharanthus roseus cells. Plant Sci 1999; 147: 177-183.

31.            Wissing JB, Kornak B, Funke A and Riedel B: Phosphatidate kinase , a novel enzyme in phospholipid metabolism : characterization of the enzyme from suspension cultured Catharanthus roseus cells. Plant Physiol 1994; 105(3): 903-909.

32.            Marfori EC, Kajiyama S, Fukusaki E and Kobayashi A: Trichosetin, a novel tetramic acid antibiotic produced in dual culture of Trichoderma herzianum and Catharanthus roseus callus. Z Naturforsch C 2002;  57: 465-470.

33.            Mitsuhashi N, Ohnishi M, Sekiguchi Y, Kwon YU, Chang YT, Chung SK, Inoue Y, Reid RJ, Yagisawa H and Mimura T: Phytic acid synthesis and vacuolar accumulation in suspension cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations. Plant Physiol 2005; 138: 1607-1614.

34.            Singh SN, Vats P, Suri S, Shyam R and Kumria MM, Ranganathan S, Sridharan K: Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in streptozotocin induced diabetic rats. J Ethnopharma 2001; 76: 269-277.

35.            Nammi S, Boini MK, Lodagala SD and Behara RB: The juice of fresh leaves of Catharanthus roseus Linn. reduces blood glucose in normal and alloxan diabetic rabbits. BMC Comp Altern Med 2003; 3: 4-7.

36.            Mostafa M, Choudhury ME, Hossain MA, Islam MZ, Islam MS and Sumon MH: Antidiabetic effects of Catharanthus roseus, Azadirachta indica, Allium sativum and Glimepride in experimentally diabetic induced rat. Bangl J Vet Med 2007; 5: 99-102.

37.            Banakar V, Malagi U and Naik R: Impact of periwinkle leaves (Catharanthus rosues) on management of diabetes mellitus. Karnataka J Agric Sci 2007; 20(1): 115-119.

38.            Natarajan A, Ahmed S, Sundaresan S, Sivaraj A, Devi K and Kumar BS: Effect of aqueous flower extract of Catharanthus roseus on alloxan induced diabetes in male albino rats. Int JPharm Sci and Drug Res 2012; 4(2): 150-153.

39.            Sharma H, Parihar L and Parihar P: Review on cancer and anticancerous properties of some medicinal plants. J Med Plants Res 2011; 5: 1818-1835.

40.            Cragg GM and Newman DJ: Plants as a source of anti-cancer agents. J Ethnopharmacol 2005; 100(1-2): 72-79.

41.            Okouneva T, Hill BT and Wilson L: The effects of Vinflunine, Vinorelbine and Vinblastine on centromere dynamics. Mol Cancer Ther 2003; 2(5): 427-436.

42.            Siddiqui MJ, Ismail Z, Aisha AF and Majid AM: Cytotoxic activity of Catharanthus roseus(Apocynaceae) crude extracts and pure compounds against Human Colorectal Carcinoma cell line. Int J Pharma 2010; 6: 43-47.

43.            Ahmad NH, Rahim RA and Mat I: Catharanthus roseus aqueous extract is cytotoxic to Jurkat Leukaemic T-cells but induces the proliferation of normal Peripheral Blood Mononuclear Cells. Trop Life Sci Res 2010; 21(2): 101–113.

44.            Wang CH, Wang GC, Wang Y, Zhang XQ, Huang XJ, Zhang DM, Chen MF and Ye WC: Cytotoxic dimeric indole alkaloids from Catharanthus roseus. Fitoterapia 2012; 83(4): 765-769.

45.            Nayak BS and Pereira LM: Catharanthus roseus flower extract has wound- healing activity in Sprague Dawley rats. BMC Comp Altern Med 2006; 6:41-46.

46.            Ramya S, Govindaraji V, Kannan KN and Jayakumararaj R: In vitro evaluation of antibacterial activity using crude extracts of Catharanthus roseus L. (G.) Don. Ethnobot Leaflets 2008; 12: 1067-1072.

47.            Goyal P, Khanna A, Chauhan A, Chauhan G and Kaushik P: In-vitro evaluation of crude extracts of Catharanthus roseus for potential antibacterial activity. Int J Green Pharm 2008; 2(3):176-181.

48.            Sathiya S, Karthikayen B, Jalil CA., Azooz MM and Iqbal M: Antibiogram of Catharanthus roseus extracts. Global J Mol Sci 2008; 3(1): 01-07.

49.            Patil PJ and Ghosh JS: Antimicrobial Activity of Catharanthus roseus – A Detailed Study. British J Pharm and Toxicology 2010; 1(1): 40-44.

50.            Wadikar MS and Nimbalkar RK: Efficacy of leaf extracts of Taphrosia purpurea and Catharanthus roseus against root rot diseases of chickpea (Cicer arietium. L.). Recent Res Sci Tech 2010; 2(7): 12-13.

51.            Sundaram R, Samuel JI and Palavesam S: In vitro antiplasmodial activity of ethanolic extracts of South Indian medicinal plants against Plasmodium falciparum. A Pacific J Trop Disease 2012; 2: 180-183.

52.            Jaleel CA, Gopi R, Lakshmanan GA and Panneerselvam R: Triadimefon induced changes in the antioxidant metabolism and ajmalicine production in Catharanthus roseus (L.) G. Don. Plant Sci 2006; 171: 271-276.

53.            Bhutkar MA and Bhise SB: Comparative studies on antioxidant properties of Catharanthus rosea and Catharanthus alba. Int J Pharm Tech Res 2011; 3(3): 1551-1556.

54.            Ara N, Rashid M and Amran MS: Comparison of hypotensive and hypolipidemic effects of Catharanthus roseus leaves extract with atenolol on adrenaline induced hypertensive rats. Pak J Pharm Sci 2009; 22(3): 267-271.

55.            Chauhan K, Sharma S, Rohatgi K and Chauhan B: Antihyperlipidemic and antioxidative efficacy of Catharanthus roseus Linn (Sadabahar) in Streptozotocin induced diabetic rats. Asian J Pharma Health Sci 2011; 2(1): 235-243.

56.            Jain A and Rawal A: Comparative study of antihelmintic activity of different extracts of Catharanthus roseus. JPRO 2011; 01(01): 23-24.

57.            Agarwal S, Jacob S, Chettri N, Bisoyi S, Tazeen A, Vedamurthy AB, Krishna V and Hoskeri HJ: Evaluation of in-vitro anthelminthic activity of Catharanthus roseus extract. Int J Pharm Sci and Drug Res 2011; 3(3): 211-213.

58.            Jyothi P and Kumara S: Central nervous system protection by Catharanthus roseus leaf extract in streptozotocin-induced diabetes in rat brain. J Pharmacognosy 2012; 3(2): 63-66.

59.            Mathur R and Chaudan S: Antifertility efficacy of Catharanthus roseus Linn: a biochemical and histological study. Acta Eur Fertil 1985; 16(3): 203-205.

60.            Gupta P: Antiestrogenic activity of petroleum ether extract of the leaves of Catharanthus roseus (Vinca rosea) in female albino mice. Asian J Exp Sci 2009; 23(1): 313-316.

61.            Murata J, Roepke J, Gordon H and Luca VD: The leaf epidermome of Catharanthus roseus reveals its biochemical specialisation. The Plant Cell 2008; 20(3): 524-542.

62.            Azimi  AA, Hashemloian BD, Ebrahimzadeh H and Majd A: High in-vitro production of anti-canceric indole alkaloids from periwinkle(Catharanthus roseus) tissue culture. Afr J Biotechnol 2008; 7(16):2834-2839.

63.            Liu DH, Jin HB, Chen YH, Cui LJ, Ren WW, Gong YF and Tang KX: Terpenoid indole alkaloids biosynthesis and metabolic engineering in Catharanthus roseus. J Int Plant Biol 2007; 49(7): 961-974.

64.            Zhao J and Verpoorte R: Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering. Phytochem Rev 2007; 6(2-3): 435-457.

65.            Guo ZG, Liu Y, Gong MZ, Chen W and Li WY: Regulation of vinblastine biosynthesis in cell suspension cultures of Catharanthus roseus. Plant Cell Tiss Organ Cult 2013; 112(1): 43-54.

66.            Kargi F, Ganapathi B and Maricic K: Indole alkaloid formation by Catharanthus roseus cells in a Biofilm reactor. Biotechnol Prog 1990; 6(4): 243-248.

67.            Archambault J, Volesky B and Kurz W: Production of indole alkaloids by surface immobilized C.roseus cells. Biotechnol Bioengg 1990; 35(7): 660-667.

68.            Cau-uitz ML, Miranda-ham J, Coello-coello B, Chi LM, Pacheco and Loyola-Vargas VM: Indole alkaloid production by transformed and non-transformed root cultures of Catharanthus roseus. In Vitro Cell Dev Biol 1994; 30(1): 84–88.

69.            Whitmer S, Canel C, Hallard D and Goncalves C, Verpoorte R: Influence of precursor availability on alkaloid accumulation by transgenic cell line of Catharanthus roseus. Plant Physiol 1998; 116(2): 853-857.

70.            Zhao J, Zhu WH, Hu Q and He XW: Improved alkaloid production in Catharanthus roseus suspension cell cultures by various chemicals. Biotechnol Letters 2000;22: 1221-1226.

71.            Xu M, Dong J and Zhu M: Effect of Nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cells. Biotechnol and Bioengg 2004; 89(3): 367-371.

72.            Srivastava NK and Srivastava AK: Influence of gibberellic acid ¹⁴CO₂ metabolism, growth, and production of alkaloids in Catharanthus roseus. Photosynthetica2007; 45(1): 156-160.

73.            Karthikeyan B, Jaleel CA, Changxing Z, Joe MM, Srimannarayan J and Deiveekasundaram  M: The effect of AM fungi and the Phosphorous level on the biomass yield and ajmalicine production in Catharanthus roseus. EurAsia J Bio Sci 2008; 2:26-33.

74.            Ramani S and Jayabaskaran C: Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light. J Mol Signalling 2008; 3: 9-14.

75.            Zhou ML, Shao JR and Tang YX: Production and metabolic engineering of terpenoid indole alkaloids in cell cultures of the medicinal plant Catharanthus roseus (L.) G. Don (Madagascar periwinkle). Biotechnol Appl Biochem 2009; 52: 313-323.

76.            Kalidass C, Mohan VR and Daniel A: Effect of auxin and cytokinin on vincristine production by callus cultures of Catharanthusroseus L. (Apocynaceae). Trop Subtrop Agroecosyst 2010; 12(2): 283-288.

77.            Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK and Yuan L: The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 2011; 157(4): 2081-2093.

78.            Wang Q, Xing S, Pan Q, Yuan F, Zhao J, Tian Y, Chen Y, Wang G and Tang K: Development of efficient Catharanthus roseus regeneration and transformation system using Agrobacterium tumifaciens and hypocotyls as explants. BMC Biotechnol 2012; 12: 34-45.

79.            Zhou ML, Zhu XM, Shao JR, Wu YM and Tang YX: A protocol for genetic transformation of Catharanthus roseus by Agrobacterium rhizogenes A4. Appl Biochem Biotechnol2012; 166(7): 1674-1684.

80.            Bacon CW and White JF: Microbial endophytes. Marcel Dekker Inc, New York,2000: 3-30.

81.            Sturz AV, Christie BR and Nowak J: Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit RevPlant Sci 2000; 19(1):1-30.

82.            Arnold E, Maynard Z, Gilbert GS, Coley PD and Kursar TA: Are tropical fungal endophytes hyperdiverse. Ecol Letters 2000; 3:267-274.

83.            Strobel G and Daisy B: Bioprospecting for microbial endophytes and their natural products. Microbiol and Mol Bio Reviews 2003; 67(4): 491-502.

84.            Kumar S and Sagar A: Microbial associates of Hippophae rhamnoides (Seabuckthorn). Plant Pathol J 2007; 6(4): 299-305.

85.            Guo B, Li H and Zhang L: Isolation of the fungus producing vinblastine. J Yunnan Univ 1998;20: 214-215.

86.            Zhang L, Guo B, Li H, Zeng S, Shao H, Gu S and Wei R: Preliminary study on the isolation of endophytic fungus of Catharanthus roseus and its fermentation to produce products of therapeutic value. Chin Tradit Herbal Drug 2000; 31(11): 805–807.

87.            Yang X, Zhang L, Guo B and Guo S: Preliminary study of a vincristine- producing endophytic fungus isolated from leaves ofCatharanthus roseus. Chin Tradit Herbal Drug 2004; 35(1): 79–81.

88.            Srinivasan K and Muthumary J: Taxol production from Pestalotiopsis sp an endophytic fungus Isolated from Catharanthus roseus. J of Ecobiotechnol 2009; 1(1): 028-031.

89.            Roy S and Banerjee D: Isolation of antimicrobial compound by endophytic bacteria from Catharanthus roseus. Int J Current Res 2010; 5: 47-51.

90.            Kafur A and Khan AB: Isolation of endophytic actinomycetes from Catharanthes roseus (L.) G. Donleaves and their antimicrobial activity.  Iranian J  Biotechnol 2011; 9(4): 302-306.

91.            Tiwari R, Awasthi A, Mall M, Shukla AK, Srinivas KS, Syamasundar KV and Kalra A: Bacterial endophyte-mediated enhancement of in planta content of key terpenoid indole alkaloids and growth parameters of Catharanthus roseus. Ind Crops and Prod 2013; 43: 306-310.

92.            Fulekar MH, Singh A, Thorat V, Kaushik CP and Eapen S: Phytoremediation of ¹³⁷Cs from low level nuclear waste using Catharanthus roseus. Indian J Pure and App Phy 2010; 48: 516-519.

How to cite this article:

Koul M, Lakra NS, Chandra R and Chandra S: Catharanthus roseus and prospects of its Endophytes: A new avenue for production of bioactive metabolites. Int J Pharm Sci Res 2013: 4(7); 2705-2716. doi: 10.13040/IJPSR. 0975-8232.4(7).2705-16

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