Enhanced Alkaloid Production from Cell Culture System of Catharanthus roseus in Combined Effect of Nutrient Salts , Sucrose and Plant Growth Regulators Malay

Callus and biomass culture of Catharanthus roseus L. were established to check for the presence of total alkaloid and its subsequent yield. Various treatments like strength of nutrient salts, sucrose concentrations and combinations of plant growth regulators (PGR’s) were applied to both MS and B5 in agar as well as suspension medium to test their effects on enhanced alkaloid content and its yield. There was no significant difference in any of the observable parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield if the culture were treated similarly in both types of media formulations (MS or B5 salts). Physical state (agar solidified or the liquid suspension) of the medium had significant effect on all the parameters particular on fresh wt, alkaloid content and yields. Although, the fresh wt. and dry wt. of biomass in suspension culture was 2-3 times less than that of callus obtained from agar medium. However, the alkaloid content and yield was 2-3 times higher in suspension culture compared to agar medium in similar treatments. The highest alkaloid content observed was 5.67mg/g dwt in B5 suspension medium containing 3% sucrose and modified with 0.5mg/l 2,4-Dichlorophenoxy acetic acid (2,4-D) + 1 mg/l Kinetin (KIN) + 2mg/l αnaphthalene acetic acid (NAA). The combined effects of these factors on the enhanced production of total alkaloids were expected to contain higher yield of anticancer vinblastine and vincristine in the cell suspension culture system. DOI : 10.14302/issn.2576-6694.jbbs-18-2475 Corresponding Author: Rajesh Kumar Srivastava, Department of Biotechnology, GITAM Institute of Technology, Gandhi Institute of Technology and Management, (GITAM) (Deemed to be University), Rushikonda, Visakhapatnam (A.P.), India. Email: rajeshksrivastava73@yahoo.co.in Running Title: Enhanced Catharanthus roseus alkaloid production


Introduction
Plants are rich source of a number of secondary metabolites including the alkaloids with an estimated count of around 3000 different types till date [1].The indole alkaloid, also known as monoterpenoid indole alkaloids (MIA) or terpenoid indole alkaloids (TIA) are derived from strictosidine which interns obtained from tryptamine and a portion from iridoid secologanin [2,3].
TIA pathway depends on indole and terpenoid precursors supplied by two convergent branches of the primary metabolites the shikimate and the isopentenyl diphosphate pathways.It involves more than twenty enzymes located in cytosol, (pro)vacuoles and chloroplasts/plastids [4].These indole alkaloids such as antimalarial quinine from Chincona officinalis, the antineoplastic camptothecine from Camptotheca acuminta, the rat poison and tonic like strychnine from Strychnos nux-vomica, antihypertensive and tranquilizer resperine from Rauvolfia sp., and the widely investigated antitumor agents vinblastine and vincristine from Catharanthus roseus are now being prescribed in modern therapeutics to treat the diseases.
Catharanthus roseus L. (Apocynacea) also known as Madagascar periwinkle is one of the most extensively used and investigated medicinal plant [5].This is a major source of the highly important anti-cancer bisindole alklaoids i.e. vinblastine and vincristine [6][7][8][9].These alkaloids are responsible for control of white cells in blood (an indicator of leukemia) thus acting as an anticancer drug by preventing mitosis in the metaphase stage when they bind to the tubulin thus inhibiting the spindle fiber formation and the cell division [10].Similarly antihypertensive alkaloids like ajmalicine and serpentine are found in the roots of these plants [11][12].Apart form these activities this plant is also found to be anti-microbial where extracts of the plant is found to be effective against S. typhi, S. aureus and B. subtilis [13][14].Other than these numerous similar pharmaceutical activities such as antidiabetic, melanomas of breast and lung, Hodgkin's and Non-Hodgkin's lymphoma are also described for this plant [15].The water extract of this plant is also known to be effective against bleeding, fever and rheumatisim [16].The anti-oxidant activity (anti-2, 2, 1-diphenyl-1-picrylhydrazyl) of this plant was also recently documented [17][18].Extensive work has been carried out on the use of this plant against a wide range of diseases [17] [19-26].Owing to very trace amount in C. roseus, has initiated widespread search for finding alternative solutions for cost effective and mass production of these drugs [7] [27-28].
Plant cell and tissue culture technique is widely used method to enhance the accumulation of such therapeutic secondary metabolites in a wide range of plants [29].The cultured plant tissues have demonstrated much higher concentrations of alkaloids than the intact plants [30][31][32].The biotechnological approaches to manipulate and regulate the production of these alkaloids of C. roseus have been overviewed recently [7].
In the present study we are applying factors such as physical state, concentrations and types of nutrient salts (MS and B5), the level of sucrose, and combinations of plant growth regulators with the aim to create stress conditions similar to that experience by plant in the field as an inductive force for enhanced production of total alkaloids containing higher amount of anticancer vincristine and vinblastine.

Selection of Plants and Explants
The Catharanthus roseus L. were grown as seedlings in the GITAM campus as hedge plant.Axillary buds shoot tips, leaf segment and roots of six week old plants growing in the GITAM campus were collected for the culture establishment.These explants were used for callus induction following standard protocol [33].Most of these explants were not successfully established due to heavy contamination by endophytic fungi (Fusarium oxysporum) and bacteria [34].Since the leaf segments were only successful in culture establishment, further studies was confined with leaf segments as explants of choice.

Sample Size
For callus culture 100ml of medium was modified for each treatment and dispensed equally in 2 culture boxes.The growth and development was measured by pooling callus from 2 culture boxes for 100ml medium, expressed in g/l and considered as first replicate.
Similarly, cell biomass from 100ml of suspension medium was pooled, expressed in g/l unit and considered as first replicate.These experiments on each treatment were repeated thrice and the results represented as mean ± standard deviations.

Callus Growth
The callus grown on agar medium was harvested after 4-weeks of sub-culture.The cell biomass from suspension culture was harvested every 2-weeks of culture/sub-culture.Callus grown in each of the culture tube or culture boxes were weighed separately.
The laboratory filter paper cut to the size of inner Petri-plate pair and dried in oven at 60°C for 2hrs to make the papers completely moisture free, cooled down to room temperature before weighing.To calculate callus fresh weight, the initial weight of dried filter paper was taken followed by weight of filter paper along with callus and represented as:

Quantitative Estimation of Total Alkaloids
The total alkaloid was estimated following the modification of protocol [33,37].A 1 mL of methanolic extract was taken and the pH brought down at 2-2.5

Alkaloid Productivity
The rate of production of alkaloid per litre per day is the productivity of system (treatment).It is

Effects of Strength and Physical State of Medium
All the observable response of fresh wt., dry wt., alkaloid content, production, productivity and the yield showed different pattern in two different physical state (agar and suspension) of the medium irrespective of MS or the B5 nutrient formulation (Table 1-4).The B5 nutrient formulation in agar solidified condition showed higher response of all the parameter compared to the MS agar medium irrespective of their strength (half, normal or one and half) (Table 1 and 3).Strength of medium (half, normal or one and half) in both the nutrient (B5 or MS) formulations under agar solidified conditions exerted significant effect with the maximum response of all the observable parameters in full strength medium and the least response in half strength medium (Table 1 and 3

Suspension Medium
The fresh and dry weight of the cell biomass was reduced 2-3 times in suspension condition when compared to agar solidified medium of both the nutrient (B5 and MS) formulation (Table 2 and 4).However, the alkaloid content, productivity and the yield were enhanced almost 2-3 time in suspension conditions as compared to agar solidified medium in their respective formulations and strength (Table 2 and 4).The total alkaloid production remained unchanged irrespective of the strength and the physical state of both (B5 and MS) the nutrient formulation.
Strength of the medium played a significant role with maximum response of all the observable parameters in normal strength and least response in half strength suspension culture (Table 2 and 4 Effect of Sucrose and PGR's

MS Agar Medium
The result of different concentrations of sucrose (1%, 3% and 5%) along with three variable combinations of PGR's in MS agar medium is shown in table 5. Multivariate analysis of variance (MANOVA) was performed and presented in table 5a.Both sucrose and PGR's significantly affect all observable parameters when analyzed alone irrespective of other factor as well as interact significantly when analyzed in combined form (Table 5a).The lower concentrations of both sucrose and PGR's were found significantly less effective than their higher concentrations.Sucrose at 3% concentrations along with PGR's combinations B (Table 5

MS Suspension Medium
The effect of different combination permutations of sucrose and PGR's in MS suspension medium on biomass and alkaloid production is presented in table 6. Multivariate analysis of variance (MANOVA) was performed for these results and presented in table 6a.Both sucrose and PGR's in MS suspension medium interacted significantly affecting all the observable parameters irrespective of whether analyzed alone or in different combinations permutations (Table 6a).In MS suspension medium fresh wt and dry wt of the cell biomass were significantly (2-3 times) less compared to agar medium.Whereas alkaloid content, productivity and the yield were 2-3 times higher than that produced in MS agar medium.The alkaloid production (in mg/l) remained unaffected irrespective to the level of sucrose or the PGR combinations in suspension medium (Table 6).The lower concentrations of both sucrose and PGR's were found least responsive than the higher concentration.All the responses were at best in the presence of 3% sucrose along with PGR's combination B (

Conclusion
In the present study we concluded that there exist a strong interaction of PGR's and sucrose The leaf explants were collected washed thoroughly under running tap water for 15 min.Surface disinfection was carried by treating with amild antiseptic solution (1% Savlon, GSK India) and 8-10 drops of Tween-20 for 5-8 min by continuous shaking.Further, surface sterilization was carried under aseptic environment over a Laminar Air Flow hood by treating first with 70% ethanol for 60 seconds followed by www.openaccesspub.orgJBBS CC-license DOI : 10.1302/issn.2576-6694.jbbs-18-2475Vol-1 Issue 4 Pg.no.-16 0.05% HgCl 2 for about 15 minutes with continuous stirring to ensure complete sterilization.Thereafter the explants were washed 3-4 times with autoclaved double distilled water to remove traces of HgCl 2 .The surface sterilized leaf explants were inoculated on the variously modified culture medium.The leaf explants were prepared either as circular disks with 1 cm diameter using a sterile cork borer or a squire segment of 1 cm 2 .Culture Medium and Culture Conditions Initially cultures were established following the standard protocols [33].At first normal strength Murashige and Skoog (MS) [35] medium containing 3 % (w/v) sucrose and different combinations of 4-D, Kinetin, 2 and NAA was for culture establishment.Later normal strength B5 culture medium [36] was modified variously for optimization.The leaf explants were treated continuously for 4-weeks in the presence of above growth regulators and each experiment was repeated three times.The leaf explants were also established on B5 medium following similar combinations of growth regulators along with 3% sucrose.To study the effect of nutrient strength on growth and multiplication of callus, biomass and alkaloid yield three different types of nutrient media (half strength, full strength and one and half strength) was prepared for both MS and B5 in agar and in suspension along with standard sucrose and PGR combination.Callus and biomass obtained from both the medium were further used for alkaloid extraction and quantification.The pH of the culture media was adjusted to 5.8 ± 0.2 prior to autoclaving (121°C, 15 min) and solidified with 1% agar (if required).The cultures were maintained at 25 ± 2 °C in an environmentally controlled air conditioned room.The culture racks were provided with a 16 h photoperiod under a photon flux density of 2,000-3,000 Lux, provided by fluorescent lamps.Induction and Multiplication of Callus and Biomass Induction of callus from leaf explants was initiated as per the standard protocol [33] with slight modifications.To test the effect of growth regulators treatments on callus induction different combinations permutations of 2, 4-D, Kinetin, NAA were used.To study the combined effect of sucrose and various PGR combinations on growth and multiplication of callus and biomass and alkaloid yield three concentrations of sucrose (1%, 3%, 5%) were used in this study in normal strength of agar as well as in suspension for both MS and B5 formulations.Three different combinations of PGR's used were (A) 0.1 mg/l of 2, 4-D + 0.5 mg/l of KIN + 1.0 mg/l of NAA (B) 0.5 mg/l of 2,4-D + 1.0 mg/l of KIN + 2.0 mg/l of NAA and (C) 1.0 mg/l of 2,4-D + 2.0 mg/l of KIN + 4.0 mg/l of NAA.).Callus and biomass obtained from both the medium were further used for alkaloid extraction and quantifications.The biomass doubling time was calculated by harvesting the suspension culture from 3 conical flasks after every 2-day interval filtered on a pre-weighed filter paper.The suspension cultures were maintained by continuous shaking at 150 RPM in a shaker incubator for 12-14 days.The suspension cultures were established and maintained for 2 cycles and sub-cultured every 12-14 days for the cell biomass harvesting and alkaloid yield.The cell biomass was harvested after 14 -days (2-weeks) by filtration on a pre-weighed filter paper (Whatman No.1).

Callus
Fresh Wt = (Weight of filter paper and the callus -Initial weight of dried filter paper) To calculate dry weight, the callus along with the filter was dried by initially at room temperature for 2 -4 days.The callus along with filter paper was further incubated at 40°C in dry heat oven for 3 -4 hours to remove the traces of moisture before weighing for dry weight.Precaution was maintained to avoid moisture exposure of petri-pales with dried callus and the filter paper.The dried callus along with the filter paper was weighed and represented as: Callus Dry Wt = (Weight of dried callus along with filter paper -Initial weight of dried filter paper) Cell Biomass Growth The cell biomass growth was calculated by harvesting the suspension culture from all 4 conical flasks of a treatment after every 2-days interval onto a pre-weighed filter paper.Filter papers (Whatman No.1) were dried in oven at 60°C for 2hrs to make the papers completely moisture free, cooled down to room temperature before weighing.One of the dried filter paper discs was used to filter simple distilled water followed by keeping over three layer of blotting paper towel for 1 hr with 2-3 changes to absorb moisture.The weight of moisture free filter paper was taken.The cell biomass were harvested by pooled filtration from 100ml medium for each treatment using the pre-weighed filter paper and kept in the petri-plates over 3 layers of blotting papers towel for absorbing of moisture by changing 2-3 times with fresh towels over a period of 1 hr.To calculate cell biomass fresh weight, the weight of moisture free filter paper along with cell biomass was taken and represented as: Biomass Fresh Wt = (Weight of moisture free filter paper and the cell biomass -Initial weight of moisture free filter paper) The cell biomass was allowed to dry at room temperature for 2-3 days by shifting on a fresh blotting papers towel every day.Finally cell biomass was dried in oven at 40°C for 2-3 hr to remove the remaining moisture before weighing for dry weight.Precaution was maintained to avoid moisture exposure of petri-pales with cell biomass.The dried cell biomass was calculated as: Biomass Dry Wt = (Weight of the dried biomass with filter paper -Weight of the dried filter paper) Extraction of Total Alkaloids About 20mg of dried callus or cell biomass was ground with 10ml of methanol in a mortar pestle, and the whole mixture kept overnight in 100ml conical flasks in a rotary shaker at 25 -50 rpm for proper mixing of the solvent.The total mixture was then centrifuged at 3000 RPM for 10 minutes and about 9.0 ml of supernatant collected.The supernatant was re-centrifuged 2-3 times until a clear supernatant was obtained.Finally 7-8ml of supernatant was collected in 25ml beakers and kept for drying at room temperature.After complete evaporation of solvent (overnight) the left over content was re-dissolved in 2ml methanol (by mixing for 1hr covered with Petri-plates) and stored in 2.0ml micro-centrifuge vials for further investigations.Alkaloid Quantifications Preparation of Standard curve for total Alkaloid Quantification The calibration curve was prepared with Bismuth nitrate pentahydrate (Bi(NO 3 ) 3 .5H 2 O) stock solution.CC-license DOI : 10.1302/issn.2576-6694.jbbs-18-2475Vol-1 Issue 4 Pg.no.-18 Bismuth nitrate stock solution was made by dissolving 10mg Bismuth nitrate and diluted with DDW (10mg of Bi (NO 3 ) 3 .5H 2 O + 5ml of Conc.HNO 3 + 95ml of DDW = 100ml of Bi(NO 3 ) 3 .5H 2 O).Series dilutions of the stock solution were made by pipetting out 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 mL stock solution into separate 10mL standard flasks and diluted to volume with double distilled water.A 1mL amount of this solution was taken, and 5 mL thiourea solution (3%) was added to it.The absorbance value of the light yellow solution was measured at 435 (430) nm against the blank containing nitric acid and thiourea.The OD value against each of the concentration is presented in figure 1.
calculated as Alkaloid Productivity (mg/l/day) = {Alkaloid Production (mg/l) / no. of days the product is harvested} Alkaloid Yield The yield of the alkaloid per treatment is represented as the percentage of alkaloid per gram dry weight of the callus or the cell biomass produced.It is calculated as Alkaloid Yield (% dwt) = {Alkaloid Content (mg/g) / 1000} x 100 Statistical Analysis The mean of three replicates of experiments, standard deviation, monovariate and multivariate analysis of variance (ANOVA and MANOVA) of results were performed by using SPSS 15 package for Window (SPSS Inc., USA) in the present study.
). Enhancing the nutrient levels to one and half strength did not improve any of the observable response.In Podophyllum peltatum tissue cultures Kadkade [42-43] showed that full strength MS medium was best suited for callus and podophyllotoxins production.Similar results were also observed by Drewes & Staden [44] for solasodine production in Solanum mauritianum.Likewise Rosli et al. [45] showed that 9-methoxycanthin-6-one production was enhanced in Eurycoma longifolia callus cultures.The concentration of basal medium was also reported to contribute towards the variation of solasodine production in callus cultures of Solanum aviculare [46].Similar findings were observed where along with auxins normal strength MS medium was found to be best for callus production of C. roseus [47].

Figure 3 .
Figure 3. Growth rate of cell biomass in 2nd and 3rd subculture cycle*.*(The normal strength MS suspension medium was added with 3% sucrose (w/v) and modified with 0.5 mg/l of 2,4-D + 1.0 mg/l of KIN + 2.0 mg/l of NAA.About 100-125 mg of friable callus (4-weeks old) was inoculated per culture vessel containing 25ml of medium.The growth of biomass in suspension culture was recorded by harvesting cultures every 2-day intervals over a period of 1 month) ). Hence it was inferred that normal strength MS medium along with standard sucrose and PGR combinations is the best combination for biomass and alkaloid yield in suspension culture.The culture production conditions of Egyptian henbane had shown that full strength MS medium as the best suited for callus and metabolites production [48].Similar results were also observed [49-51] for alkaloid production in Holarrhena antidysenterica to study the effect of biochemical engineering on secondary metabolites derived from plant source by implementation of biochemical engineering production using cell suspension culture.MS suspension culture at normal strength proved to be the best nutrient source for biomass and alkaloid production [52] while studying the production of some important secondary metabolites from medicinal plants by plant tissue cultures.These findings corroborated with the findings where cytokinin in B5 media was found to very helpful in increasing the total alkaloid content in Catahranthus roseus [53-54].Hence, it was inferred that both normal strength MS and B5 suspension culture along with standard sucrose and PGR combinations as the best combination with better response of biomass and alkaloid yield in B5 suspension culture.Similar finding has proved that Catharanthus roseus biomass when transferred to B5 suspension medium showed extensive alkaloid production when used as immobilized cells [55-56].
concentrations for the induction, growth and multiplication of leaf derived callus in both MS agar and suspension medium.Further, the physical state of the medium (agar solidified and liquid suspension) played an important role on all the observable parameter of fresh wt and dry wt as well as alkaloid content, production, productivity and yield.The standard culture conditions induced considerable amount of variations in total alkaloid production from callus grown on agar medium and the cell biomass in suspension of Catharanthus roseus.Different factors such as concentrations of nutrient salts and sucrose used as carbon source along with the plant growth regulators tested in two media formulations (MS and B5 salts) significantly effected all the parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield irrespective of whether analyzed alone or in combinations.The yield of total alkaloid was optimized for enhanced production in different combinations permutations of nutrient salts, sucrose and plant growth regulators.There was considerably enhanced amount (5.67 mg/g dwt) of total alkaloid yield in the present study similar to that observed in other reports [61-65].The obtained results need to be exploited further for separation and purification of different indole alkaloids to test their pharmacological activities.

Table 1 .
Effect of Nutrient Strength on Growth and Multiplication of Callus and Alkaloid in MS Agar Medium*.

Table 2 .
Effects of Nutrient Strength on Biomass and Alkaloid in MS Suspension Culture*.

Table 3 .
Effect of Nutrient Strength on Growth and Multiplication of Callus and Alkaloid in B5 Agar *(Agar (1%) solidified B5 medium prepared in the above strength (concentrations) were added with 3% sucrose (w/v) and modified with 0.5 mg/l of 2,4-D + 1.0 mg/l of KIN + 2.0 mg/l of NAA.About 100-150 mg of callus was inoculated per culture vessel and the growth was recorded after 4-weeks of subculture.)

Table 4 .
Effects of Nutrient Strength on Biomass and Alkaloid in B5 Suspension Culture*.

Table 5a .
Multivariate Analysis of Variance: Tests of Between-Subjects Effects of different Concentrations of Sucrose and PGR's on Callus and Alkaloid in MS Agar Medium.

Table 6 .
Effects of Sucrose and PGR's Concentrations on Biomass and Alkaloid in MS Suspension Medium*.

Table 6a .
Multivariate Analysis of Variance: Tests of Between-Subjects Effects of different Concentrations of Sucrose and PGR's on Biomass and Alkaloid in MS Suspension Medium.