THE EFFECT OF ELICITORS ON THE PRODUCTION OF VASICINE FROM JUSTICIA ADHATODA L. CELL SUSPENSION CULTURES

THE EFFECT OF ELICITORS ON THE PRODUCTION OF VASICINE FROM JUSTICIA ADHATODA L. CELL SUSPENSION CULTURES

Rashmi Pa and Linu Mathew*

School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, 686 560, India

ABSTRACT

In this study, α-naphthyl acetic acid and mannitol were used as elicitors to improve the productivity of useful metabolite, vasicine for archiving high concentration in Justicia adhatoda L. cell suspension cultures. When the cells were treated with 3mg 100 ml^-1 suspension of α-naphthyl acetic acid,   the vasicine concentration was increased by 1.65 mg ml^-1 in 15 days culture compared to untreated cells. 0.32M 100ml^-1 mannitol could also promote the synthesis of vasicine in the cell cultures. After 15 days of the cell culture, it was found that the vasicine concentration was 1.48 mg ml^-1 more than the control cell cultures.

Mannitol, Justicia adhatoda L.

INTRODUCTION: Plant cell culture is often an effective system to study the biological significance of bioactive metabolites under in vitro conditions, as well as for producing natural products for bioprocessing applications ¹. It has been industrially used for the synthesis of secondary metabolites ². The regular increase in demand in world market for natural, renewable products has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products, and has paved the way for new research exploring secondary product expression in vitro ³.

Plants express a number of morphological and physiological responses to a range of physical and chemical factors from the environment, known as elicitors, which activate chemical defenses in plants ⁴. The synthesis of plant secondary metabolites can be stimulated by acting on different parameters: environmental factors, use of precursors of the targeted molecules, use of elicitors and, genetic transformation of the plant ⁵. Secondary metabolites production in entire plants depends on endogenous ⁶ and exogenous factors ⁷.

Enhancement of secondary metabolites by elicitor application is one of the few strategies which have recently found commercial application. Elicitors can be abiotic or biotic compounds of mainly microbial origin or non-biological origin, which upon contact with higher plant cells; trigger the increased production of pigments, flavors, phytoalexins and other defense related compounds ⁸.

The recent development of elicitation has opened a new avenue for the production of secondary compounds. Secondary metabolite synthesis and accumulation in cell cultures can be triggered by the application of elicitors to the culture medium ⁹.

Vasicine produced by Justicia adhatoda is used for the treatment of various diseases and disorders, mostly for respiratory tract ailments. But the natural production of vasicine is insufficient to fulfill the requirements. This work reports the successful approach for the elevated production of vasicine by cell suspension cultures after using α- naphthyl acetic acid (NAA) and mannitol as elicitors.

Present study was conducted to examine the effect of elicitors (α-naphthyl acetic acid (NAA) and mannitol) on the production of vasicine in the cell suspension cultures of J. adhatoda. The plant shows wide seasonal variation in vasicine content in its leaves. It exhibited higher levels of vasicine twice in a year i.e. 3.0% in March and 1.4% in September. Interestingly, it coincided with the flowering of the plant. In March, it was full bloom condition and in September, it was partial flowering. During the vegetative stage, the plant contained very low concentration of vasicine ¹⁰. The yield of the vasicine from different samples in India ranged from 0.541 to 1.105% on dry basis. Yield as high as 2.18% on dry basis has been reported from a foreign sample of which more than half was the l -form and the remainder the dl-form of the alkaloid ¹¹.

The present study mainly aimed to analyze the effect α-naphthyl acetic acid and mannitol, as elicitors on the production of vasicine from cell suspension cultures of J. adhatoda L.

MATERIALS AND METHODS:

Suspension Culture: In order to initiate suspension culture of J. adhatoda, 50gm of friable calli from culture tubes were transferred to 250 ml Erlenmeyer flasks containing 100 ml of MS medium with 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg of NAA. And another set of suspension culture with mannitol of 0.08 M, 0.16 M, 0.32 M and 0.64 M (E. Merck India) were also prepared. 250 ml Erlenmeyer flasks containing 100 ml of MS medium with 50gm of friable calli, without elicitors, was kept as control. The flasks were incubated on a rotary shaker at 120 rpm using the same culture conditions used for callus culture.

Extraction of Secondary Metabolite from Suspension Culture: From the suspension culture of friable calli, the supernatant was collected and extracted with methanol for 30 minutes by using a separating funnel. The extracts collected and concentrated to half the volume.

Quantification of Vasicine: Quantification of total alkaloids was done by spectrophotometric method with tropaeolin ‘OO’ (SIGMA ALDRICH). Colored complex developed was measured at 545 nm against blank. The amount of total alkaloids in the samples was calculated using standard curve of vasicine (SPIC India Ltd, Chennai). The content of the total alkaloids was expressed as vasicine ^{12, 13}.

All the procedures were done in three replicates

RESULT: Vasicine content was found to be 6.73mg ml^-1 and 6.56 mg ml^-1 in suspension cultures (Table 1 and 2) with 3mg 100 ml^-1α-Naphthyl acetic acid and                          0.32M 100ml^-1 Mannitol respectively which were higher than that of field grown plants ^{12, 13}  and control culture (5.08mg ml^-1) (Table 3).

TABLE 1: INFLUENCE OF NAA AS ELICITOR FOR ENHANCING THE PRODUCTION OF VASICINE IN SUSPENSION CULTURES OF J. ADHATODA

TABLE 2: INFLUENCE OF MANNITOL AS ELICITOR FOR ENHANCING THE PRODUCTION OF VASICINE IN SUSPENSION CULTURES OF J. ADHATODA

 TABLE 3: PRODUCTION OF VASICINE IN SUSPENSION CULTURES OF J. ADHATODA WITHOUT AN ELICITOR (CONTROL)

All the observations strongly support the use of α-naphthyl acetic acid and Mannitol as elicitors for vasicine production from J. adhatoda. But after the 20^th day of inoculation, a slight reduction in the concentration of vasicine was noticed (Figure1 and 2) and in the culture without elicitors, the reduction was detected after the 15^th day of inoculation.

FIGURE 1: EFFECT OF NAA AS ELICITOR FOR VASICINE PRODUCTION FROM J. ADHATODA SUSPENSION CULTURE WITH CONTROL

FIGURE 2: EFFECT OF MANNITOL AS ELICITOR FOR VASICINE PRODUCTION FROM J. ADHATODA SUSPENSION CULTURE WITH CONTROL

DISCUSSION: Vasicine is one of the major alkaloid produced by J. adhatoda. It has great importance in Ayurveda due to its bronchodilatory effect. It also has anti-inflammatory, anti-bacterial activity ¹⁴, anticestodal activity ¹⁵, antileishmanial activity ¹⁶ and anti-helminthic activity ¹⁷.

Mannitol is a polyol normally synthesized in numerous species of vascular plants, which serves as an alternate metabolic reserve as well as an osmoprotectant. Mannitol accumulation increases when plants are exposed to a low water potential and the most obvious function of this compound is in osmotic adjustment.

Therefore, mannitol has been proposed to enhance tolerance to water deficit stress primarily through osmotic adjustment ¹⁸. It is already described the role of mannitol as hydroxyl radicals scavenger.

The mannitol added to cultures causes a mild osmotic stress to cell cultures and, in time, this treatment may activate an adaptive mechanism that can render the cells tolerant to more severe treatments. On a medium supplemented mannitol, the cultured cells take up mannitol that can act as an osmoprotectant. However, accumulation of mannitol may have adverse effects, even though mannitol is a compatible solute, the target plant may not tolerate high levels of mannitol ¹⁹.

According to ^{10, 11}, J. adhatoda showed seasonal variation and the vasicine content was high in dry climate. Hence the results strongly supported addition of mannitol as elicitor for vasicine production. NAA is also act as a good elicitor and support the production of vasicine.

In the present work, the cell cultures showed great sensitivity towards elicitors by producing vasicine when supplemented with NAA and Mannitol at concentration as 3mg 100ml^-1 and 0.32M 100ml^-1 respectively. The increased production, through elicitation, of the secondary metabolites from plant cell culture has opened up a new area of research, which could have important economic benefits for bio industry. Elicitation studies have shown promise in increasing yields and cutting production costs.

REFERENCE:

1. Walker TS, Bais HP and Vivanco JM: Jasmonic Acid-Induced Hypericin Production in Cell Suspension Cultures of Hypericum perforatum (St. John’s Wort). Phytochemistry 2002; 60:289–293.
2. Riedel H, Akumo DN, Thaw Saw NMM, Kütük O, Neubauer P and Smetanska I: Elicitation and Precursor Feeding Influence Phenolic Acids Composition in Vitis vinifera Suspension Culture. African Journal of Biotechnology 2012; 11(12):3000-3008.
3. Anand S: Various Approaches for Secondary Metabolite Production through Plant Tissue Culture.Pharmacia 2010; 1:1-7
4. Bota C and Deliu C: The Effect of Copper Sulphate on theProduction of Flavonoids in Digitalis lanata Farmacia 2011; 59(1):113.
5. Vu TD, Tran  TLM, Biteau  F, Mignard B, Fèvre JP, Guckert A, Bourgaud F and  Gontier etE: Improvement of Secondary Metabolites Production in Hydroponic Cultures by Mechanical and Biological Processes. Proceedings of International Workshop on Biotechnology in Agriculture, Nong Lam University Ho Chi Minh City October 20-21 2006; 195-200.
6. Hashimoto T, Hayash A, Amano Y, Kohno J, Iwanari H, Usuda S and Yamada Y: Hyoscyamine 6-Beta-Hydroxylase, an Enzyme Involved in Tropane Alkaloid Biosynthesis, is localized at the Pericycle of the Root. J. Biol. Chem. 1991; 266:4648-4653.
7. Boitel-Conti M, Laberche JC, Lanoue A, Ducrocq C and Sangwan-Norreel BS: Influence of Feeding Precursors on Tropane Alkaloid Production during an Abiotic Stress in Datura innoxia Transformed Roots. Plant Cell Tissue and Organ Culture 2000; 60:131-137.
8. Pirian K and Piri K: Effect of Methyl Jasmonate and Salicylic Acid on Noradrenalin Accumulation in Hairy Roots of Portulaca oleracea International Research Journal of Applied and Basic Sciences 2012; 3 (1):213-218.
9. Subathra DC and Mohana SV: In Vitro Studies on Stimulation of Gymnemic Acid Production Using Fungal Elicitor in Suspension and Bioreactor Based Cell Cultures of Gymnema sylvestre Br. Recent Research in Science and Technology 2011; 3(4):101-104
10. Bagchi, GD, Dwivedi PD, Haider F, Singh S, Srivastava S and Chattopadhyay SK: Seasonal Variation in Vasicine Content in Adhatoda Species Grown Under North Indian Plain Conditions. J. Med. Aromatic Plant Sci. 2003; 25:37-40.
11. Arambewela LSR, Ratnayake CK and Jayasekera JS: Vasicine Contents and their Seasonal Variation in Adhatoda vasica. Fitoterapia 1988; 59:151-153.
12. Soni S, SheetalA, Patel G and Rajani M: Validation of Different Methods of Preparation of Justicia adhatoda Leaf Juice by Quantification of Total Alkaloids and Vasicine. Indian J Pharm Sci 2008; 70(1):36–42.
13. Rashmi PA, Reshma J and Linu M: Isolation and characterisation of vasicine from in vitro cultures of Justicia adhatoda L. Int J Pharm Bio Sci 2012; 3(3):B58 – 64.
14. Karthikeyan A, Shanthi V and Nagasathaya A: Preliminary phytochemical and Antibacterial Screening of Crude Extract of the Leaf of Justicia adhatoda. L. Int. J. Green Pharm. 2009; 3:78-80.
15. Yadav AK and Tangpu V: Anticestodal Activities of Justicia adhatoda Extract against Hymenolepis diminuta Infections in Rats. J. Ethnopharmacol 2008; 119:322-324.
16. Khaliq T, Misra P, and Gupta S: Peganine Hydrochloride Dihydrate an Orally Active Antileishmanial Agent. Bioorg. Med. Chem. Lett.2009; 19:2585-2586.
17. Al-Shaibani IRM, Phulan MS and Arijo A: Ovicidal and Larvicidal Properties of Justicia adhatoda (L.) Extracts against Gastrointestinal Nematodes of Sheep in vitro. Pakistan vet. J. 2009; 28:79-83.
18. Loester WH, Tyson RH, Everard JD, Redgwell RJ and Bieleski RL: MannitolSynthesis in higher plants: evidence for the role and characterization of a NADPH- dependent Mannose - 6-phosphatereductase. Plant Physiol 1992; 98:1396- 1402.
19. Mitoi EM, Holobiuc I and Blindu R: The Effect of Mannitol on Antioxidative Enzymes in vitro Long Term Cultures of Dianthus tenuifolius and Dianthus spiculifolius. J. Biol. - Plant Biol2009; 54 (1):25-33.
    

    ---

    How to cite this article:

    Pa R and Mathew L: The Effect of Eelicitors on the production of Vasicine from Justicia adhatoda L. Cell suspension cultures. Int J Pharm Sci Res. 3(10); 3923-3926.

    ---