Cell Suspension Culture of Plumbago europaea L. Towards Production of Plumbagin.

BACKGROUND: Plumbagin is as an important bioactive secondary metabolite found in the roots of Plumbago spp. The only one species, Plumbago europaea L., grows wild in Iran. The therapeutic use of plumbagin is limited due to its insufficient supply from the natural sources as the plants grow slowly and take several years to produce quality roots.
OBJECTIVES: To develop an efficient protocol for the establishment of callus and cell suspension cultures of P. europaea and to evaluate production of plumbagin in callus and cell suspension cultures of P. europaea for the first time.
MATERIAL AND METHODS: Stems and leaves explants were cultured on agar solidified (7% w/v) MS media, supplemented with different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction. The rapid growing calli were cultured in liquid Murashige and Skoog (MS) media in agitated condition for establishing cell suspension cultures of P. europaea. Moreover, the effects of light and dark conditions on the cell growth, cell viability and plumbagin production in cell suspension cultures of P. europaea were assessed.
RESULTS: Friable calli were successfully induced using stem segments of P. europaea in semisolid MS medium supplemented with 1 mg.L-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg.L-1of kinetin (Kin). Optimal cell growth was obtained when the cells were grown in MS liquid media supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 kinetin with an initial cell density of ~3×105 cells per ml incubated in the dark at 25 ± 1 °C. Growth curve revealed that the maximum cell growth rate (14.83×105 cells per ml) achieved on the day 18 and the highest plumbagin content (0.9 mg.g-1 Dry Cell Weight (DCW)) in the cells was obtained at the late exponential phase under dark condition which determined by High Performance Liquid Chromatography (HPLC) technique. Based on the obtained results, cell viability remained around 82.73% during the 18 days of cell culture in darkness. These suspension cultures showed continuous and stable production of plumbagin.
CONCLUSIONS: Our study suggests that cell suspension cultures of P. europaea represent an effective system for biosynthesis and production of plumbagin as a valuable bioactive compound.

1. Background

Plumbago L. (Plumbaginaceae) encompasses 12 species which are found mainly in Europe, the Mediterranean region, north Africa and southwest Asia. Among the species, only Plumbago europaea L. grows wild in Iran (2). The roots of P. europaea are reported to be the main source of plumbagin (5-hydroxy, 2-methyl, 1-4 naphthoquinone) as an important bioactive secondary metabolite (3, 4). Plumbagin possesses a wide range of pharmacological activities such as anticancer, antimicrobial, antimalarial, insecticidal, antiatherosclerosis, antioxidant, antifertility, filaricidal and cardiotonic agents (5-15). The quantity of plumbagin varies depending on growth and developmental stage of the plant as well as its locality and season conditions (16). Plumbagin has been shown to inhibit cell proliferation by inducing cells to undergo autophagic cell death (17, 18). According to Sandur et al (2006), Plumbagin down-regulates the expression of NF-κB regulated anti apoptotic, proliferative and angiogenic gene products, which leads to apoptosis (19).

2. Objective

The biosynthesis of plumbagin and other naphthoquinones such as isoshinanolone and droserone takes place through the acetate-malonate pathway (20). The commercial use of wild grown plants has led to their rapid decline in the natural environment. On the other hand, the production of plumbagin by chemical synthesis is not commercially promising (21, 22). Fieser and Dunn (1936) successfully synthesized plumbagin entirely through chemical process but the synthetic approach of plumbagin production was not found to be commercially promising by Ichihara et al (1980) and Wurm et al (1986) (21-23). Thus, it is important to develop an efficient biotechnological system to enhance plumbagin production. Although biotechnological production of plumbagin using plant cell cultures of some Plumbago species has been studied (2, 24), the yields are required to be considerably enhanced for possible commercial exploitation. Light is an important factor, which affects not only growth and development of the plant but also the biosynthesis of primary and secondary metabolites (25, 26). Cell suspension culture is the preferred and reliable approach since it gives a homogenous fast growing material and is easy to scale-up. The large-scale cultivation system of cell and tissue culture using bioreactor is promising for the production of plant biomass and secondary metabolites. It has several advantages such as large mass propagation, controlled environment, consistency of the product and reducing the micropropagation cost (27). The present study introduces the capability of the cell cultures of P. europaea for in vitro biosynthesis of plumbagin and provides a framework for further investigations.

3. Materials and Methods

3.1. Plant Materials

The seeds and fresh samples of intact roots of Plumbago europaea were obtained from Research Institute of Forests and Rangelands, Tabriz, Iran. The surface disinfection of seeds was carried out under a laminar flow cabinet using sulfuric acid (98%) for 5 min, ethanol (70 %) for 45 s and sodium hypochlorite solution (2 %) for 10 min. Then, they were rinsed in sterile distilled water and cultured on the solidified hormone-free MS media (28). The media were incubated in a phytotron at 25 ± 1 °C under 16/8 h light/dark photoperiod (with cool white fluorescent light (40µmol m-2 s-1)). After three weeks of culture, in vitro grown plantlets were used as a source of explants ().

3.2. Induction of Callus

Stems and leaves segments (1-1.5 cm) were cultured on agar solidified (7% w/v) MS media, supplemented with the different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction ( and ). The pH of the medium was adjusted to 5.8 before autoclaving. The media were autoclaved at 121 °C, ~105 kPa for 20 min. All cultures were incubated in dark at 25 ± 1 °C and relative humidity maintained at 80%. They were sub-cultured every 28 days. Thereafter, callus induction percentage, fresh weight of callus and texture of callus were evaluated using a completely randomized design method. Five replicates and five explants in each replicate were used. After 3 sub-cultures, the friable calli tissues were transferred to liquid MS media.

3.3. Establishment of Cell Suspension Cultures

Cell suspension cultures were developed by transferring 3g of friable calli derived from stem explants into 250 ml flasks containing 100 ml of fresh liquid MS medium supplemented with 2, 4-D (1 mg.L-1), Kin (0.5 mg.L-1) and sucrose (30 g.L-1). The pH of the media was adjusted to 5.8 before autoclaving. The suspension cultures were incubated at 25 ± 1 °C under 16 h light: 8 h dark photoperiod of cool white fluorescent light (40µmol m-2 s-1) or dark condition with continuous shaking at 110 rpm. Sub-culturing cell suspensions were conducted every 2 weeks by transferring an initial cell density of 3×105 cells per ml to the same liquid medium. To establish the growth kinetics and plumbagin production, individual flasks were sacrificed every 3 days over a 27 day period and used to determine the number of cells, cell viability and plumbagin content. Readings were carried out from three flasks for each parameter.

3.4. Cell Number

Cell density was estimated and monitored using a hemocytometer according to the method described by Oropeza et al. 2001 (29).

3.5. Cell Viability

The cell viability assay was determined by staining the cells using 0.25 % Evan’s blue by the method described by Rodrı´guez-Monroy and Galindo 1999 (30).

3.6. Assay of Plumbagin

A quantity of 1g dried samples subjected to cold extraction using 100 ml chloroform for 72 h. The supernatant was collected by decantation into an amber colored bottle. The crude extracts were filtered by filter paper (Whatman, no. 1), evaporated by the rotary evaporator, dissolved separately in 5 ml methanol and filtered through 0.45 µm membranes before injection to the HPLC. A Breeze HPLC system from Waters Corporation (USA) was used at a wavelength of 270 nm with the outputs to record and analyze using a software (Breeze, USA). Plumbagin as standard was purchased from Sigma-Aldrich. An amount of 50 µL of methanolic extract of sample was injected into C8 analytical column (250 mm * 4.6 mm, particle size 5 µm; Perfectsill, MZ-Analysentechnik, Germany). The mobile phase was a mixture of methanol: water (80:20) and run at the isocratic condition with a flow rate of 0.9 ml min-1. The chromatographic peak of plumbagin was confirmed by comparing its retention time with that of the standard (31). Quantitative estimation of plumbagin was carried out based on the peak area of specific concentrations of the sample and the standard. The area under the peaks of plumbagin was integrated and converted to concentration using its calibration curve.

3.7. Statistical Analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test for inter-group comparisons, using the SPSS 16.0 (Statistical Program for Social Sciences) program. The level of significance was set at P < 0.05.

4. Results

4.1. Callus Induction

and show the effects of different combinations of plant growth regulators tested for callus induction, callus fresh weight and nature of callus in the stem and leaf explant. Callus tissues were effectively induced in leaf and shoot explant by all combinations of 2, 4-D and Kin or BA ( and ) whereas no callus was generated on growth regulator free MS media. Therefore, it can be concluded that callus induction from leaf and stem explants of P. europeae is mainly influenced by the exogenous plant growth regulators. In stem segments, callus induction was mostly appeared on days 6-9 of culture, whereas in the leaf explants, callus induction occurred slower and after 11-13 days. In the present study, the cytokinin (BA or Kin) treatment without auxin (2, 4-D) did not induce callus, whereas the exclusive presence of 2, 4-D in the medium and the absence of cytokinins (BA or Kin), was resulted in higher callus weight ( and ). However, the combinations of auxins (2, 4-D) and cytokinins (BA or Kin) were found to produce more callus tissues than the media containing auxin or cytokinin alone.

Using stem explants, the highest callus fresh weight (253.20 mg FW) and the best callus texture were obtained in MS medium containing 1 mg.L-1 2, 4-D and 0.5 mg.L-1 Kin on the 28th day and were yellowish-white in color and more friable in nature (). After 4 weeks, the calli were sub-cultured on the same medium, to achieve more friable callus prior to initiation of cell suspension cultures. In comparison, MS medium supplemented with 1 mg.L-1 2, 4-D and 1 mg.L-1 Kin was suitable for callus development from leaf explants (179.10 mg FW) (). In the callus culture (stem-derived ) grown on MS medium supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 Kin after 3 sub-cultures, plumbagin content was 0.084 mg.g-1 DW (). We found also that the intact roots of P. europaea in natural environment contain 1.78 mg.g-1 DW of plumbagin (). Stem explants exhibited the higher frequency of callus formation and the higher fresh weight compared to leaf explants. Hence, stem explants were used for further experiments.

4.2. Suspension Cultures

Suspension cultures were initiated by transferring fresh friable stem-derived calli into liquid MS medium supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 Kin. The suspension cultures were established by regular sub-culturing and became completely uniform after 6 weeks. Cell growth was measured by recording the cell number every 3 days. In this study, we compared the influence of lighting conditions (total darkness versus 16 h light: 8 h dark photoperiod) on the cell growth, cell viability and plumbagin production of cell suspensions of P. europaea. shows the effect of light and dark conditions (total darkness versus 16 h light: 8 h dark photoperiod) on the growth of cell cultures and plumbagin accumulation.

In a suspension culture of P. europaea, an S-shaped growth-curve was generally observed in both lighting conditions ( and 3B). Cell suspensions were characterized by an initial lag phase around day 6 (a period of cellular adaptation to the new medium). The initial lag phase was followed by exponential growth phase from 6th to 18th day of culture. The cultures showed about 5-fold increase in cell density (14.83×105 cells per ml) and 4-fold increase in cell density (11.97×105 cells per ml) on the 18th day under dark and light conditions, respectively. The stationary phase followed by a gradual reduction in cell density due to senescence of the cells and deficiency of the nutrient in the medium (). Based on the obtained growth curve, the day of the sub-culture was optimized between days 15 and 18 of incubation, which was the end of the exponential growth phase according to the suggestion of Stafford and Warren (32).

Our results indicated that the optimal cell growth was obtained when the cells were grown in MS media supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 of Kin with an initial cell density of ~3×105 cells per ml in dark. The results showed that cell growth requires a certain initial density of cells. Poor results obtained at low (1×105 cells per ml) and high (6×105 cells per ml) initial cell density (data not shown). The present study confirmed the concept that the stimulatory influence of initial inoculums affects the cell growth kinetics in plant cell cultures (33).

Microscopic observation of suspension cultures showed that the cells were round or oval shape (). Based on the Evans blue staining method, the cell viability was calculated under a light microscope (staining the dead cells) (). Cell viability of the cultures under regular photoperiod condition did not show any significant difference with those in darkness. The survival rate at the 18th day for cells under light was 80.8%, while under the dark condition it was about 82.73% (). The viability of cells decreased slightly until the 18th day (82.73%) and consequently decreased by 56.4% on the 27th day in the dark. ().

Dynamics of plumbagin accumulation in P. europaea suspension culture, during its cultivation cycle presented in . Our data revealed that the dynamics of plumbagin production in P. europaea suspension culture was growth-associated during the growth cycle (). Intriguingly, plumbagin accumulation was high in the late logarithmic phase (). The maximum contents of plumbagin in cell suspension cultures incubated in dark (0.9 mg.g-1 DCW) and in photoperiod condition (0.52 mg.g-1 DCW) were observed when the cultures moved from the exponential into the stationary phase (at the 21st day of cultivation cycle).

5. Discussion

Callus initiation is a primary and necessary step in a number of tissue culture processes such as establishment of cell suspension cultures, regeneration of plants and indirect somatic embryogenesis (34-36). In the current study, callus culture was used for establishment of cell suspension cultures of P. europaea for the first time. To obtain a good suspension culture, it is critical to initiate the suspension cultures from a friable callus source (37, 38). Using leaf and stem explants callogenesis was successfully achieved in both types of the explants. The callus obtained from stem explants in MS medium containing 2, 4-D (1 mg.L-1) and kinetin (0.5 mg.L-1) exhibited highest fresh weight compared to leaf explants and produced soft and friable. However, stem explants were more suitable for callus induction than leaf explants. Similar findings were reported for Corydalis saxicola (39) and Orthosiphon stamineus (40). Seeni and Komaraiah (2002) have used stem derived callus for the establishment of cell suspension cultures of P. indica.

In the present study, MS basal liquid medium supplemented with 3% sucrose and 1 mg.L-1 (2, 4-D) and 0.5 mg.L-1 BA, was found producing higher biomass accumulation of (253.20 mg FW) on day 28 (). Our results exhibited that callus induction from leaf and stem explants of P. europeae is mainly influenced by the exogenous plant growth regulators. However, the combinations of auxins and cytokinins were found to produce more callus tissues than the media containing auxin or cytokinin alone. The positive effect of plant growth regulators on callus induction has been previously reported for several plant species (35, 41). In fact, auxins play a more important role in callus induction and cytokinins (BA or Kin) facilitate their effects (39, 42, 43). Synergetic effects of plant growth regulators play a critical role in callus induction and cell differentiation (44). Auxins and cytokinins are necessary for cell division at the G1-S and G2-M transitions in cultured plant cells and in planta (45). Auxins stimulate the acidification of the cell wall resulting in increasing extensibility and also induce the transcription of specific mRNAs which code for proteins associated with cellular growth. In comparison, cytokinins act directly on the cell cycle by regulating the synthesis of proteins involved in the formation and operation of the mitotic spindle (45-47).

Our results indicate that the combination of 2, 4-D and Kin was more effective than 2, 4-D and BA to induce adequate calli from stem explants of P. europaea. This finding is in fair agreement with previous reports in callus induction of P. indicia showing the noteworthy effect of 2, 4-D and Kin (24, 48). In general, our findings indicated that the type and combination of plant growth regulators and explant types had significant effects on callus induction of P. europaea. These differences can be attributed to variation in endogenous hormone content in the explant or different sensitivities of the tissue to the plant growth regulators (49).

Cell cultures of P. europeaa showed a sigmoid growth pattern, which started with the lag phase, followed by the exponential phase and ended with the stationary phase. Hence, subcultures have to be carried out before the cultures enter the stationary phase in order to maintain the cell lines for prolonged durations and in a healthy condition. Similar results were previously reported in the establishment of suspension cultures of P. indica (2, 24). It seems that consumption of the nutrients and other restricting factors in the medium has an influence not only on the growth and cell viability but also on the synthesis of this important secondary metabolite during the stationary phase. It was previously revealed that in around 50% cell viability, the establishment of suspension cultures will fail (50). Our results showed that the cell suspension cultures of P. europaea have successfully established.

Light is an important physical factor which affects not only growth and development of the plant but also biosynthesis metabolites in many plant cell cultures (25, 26). Also, the effects of light and dark on cell growth, plumbagin formation and cell viability were examined by cell suspension culture of P. europeaa in this study. Cell cultures grown in dark exhibited higher cell growth (14.83×105 cells per ml) at the end of exponential phase compared to cell cultures under the light condition (11.97×105 cells per ml) ( and 3B). The plumbagin content in cell suspension cultures incubated under dark condition was 1.7-fold higher than in cell suspension cultures grown under photoperiod condition. Bias et al. stated that the cell cultures of Hypericum perforatum incubated in darkness showed an increase in biomass yield and hypericin accumulation (51). Our findings confirm the previous reports showing that light has an inhibitory effect on the accumulation of some secondary metabolites such as shikonin and nicotine in the hairy root and cell suspension cultures of Lithospermum erythrorhizon (52, 53) and phenolic compounds in cell suspension cultures of Thevetia peruviana (54).

The results revealed that the dynamics of plumbagin production in P. europaea suspension culture was growth-associated during the growth cycle in both lighting conditions (). Our findings are in agree with the previous reports of Komaraiah, Ramakrishna (2) and Satheeshkumar, seeni (24), who shown that increase in the concentration of plumbagin can be parallel to the growth of cells in cell suspension cultures.

Production of plumbagin in cell suspension cultures has been investigated earlier in Dionaea muscipula, Drosera species (55) and Drosophyllum lusitanicum (56). Plumbagin was also isolated from cell suspension culture of P. rosea (2). Satheeshkumar and seeni have reported 0.028 mg.g-1 DW and 0.05 mg.g-1 DW plumbagin in cell suspension cultures and callus cultures of P. indica (24). Heble et al. (1974) reported that plumbagin concentration was 0.0001-0.0003% FW callus culture in P. zeylanica. The amount of plumbagin in root cultures of P. rosea was 0.016 ± 0.0030% DW (57). Higher plumbagin production (4.3 mg.g-1) was reported in suspension cultures of P. indica (2). The contradictory results reported by several authors concerning the accumulation of plumbagin in Plumbago cell and callus cultures may be explained by the variation in nutrient media and culture conditions used for initiation of Plumbago cell suspension, difference in genotype of explants and the unstable level of these compounds during culture.

The plumbagin content in intact roots of P. europaea in the natural environment was 1.78 mg.g-1 DW. Plumbagin production in this tissue was 2- and 22-folds higher than in cell cultures grown in the dark (0.9 mg.g-1 DCW) and callus culture (0.084 mg.g-1 DW), respectively. Our results are in corroboration with Nahahlka et al. (1996) who has also reported more amounts of plumbagin in Drosophyllum suspension cultures than in callus. Although the concentration of plumbagin in callus and suspension cultures was less than the intact roots, the growing time of suspension culture is very shorter in comparison to field growing plants. Furthermore, plant cell suspension culture is the most favorite technique, since it gives a homogenous fast growing material, represents simple methods and is easy to scale-up. Using this technique, the dependence on intact plants and the mass destruction of natural resources will be reduced. However, further investigations are needed to improve plumbagin production under in vitro condition.

6. Conclusions

In the present study, we established a reliable and effective callus and cell suspension cultures of P. europaea. Further, the obtained data revealed that plumbagin could produce through callus and cell suspension cultures of P. europaea. The friable callus tissues were produced by culturing the stem explants on MS medium supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 Kin. The ability of cell suspension cultures to produce plumbagin was examined during their growth cycles. Cell growth and plumbagin content were higher when cultures were incubated in the dark than those cultures under a photoperiod mode. This study showed that suspension cultures of P. europaea as a green factory is a promising technology for the synthesis of the valuable compound plumbagin.

Table 1.

Effect of the exogenous plant growth regulators on the callus induction of internodal explants of Plumbago europeae after 28 day.

Auxin, mg/l	Cytokinin, mg/l	Callus induction rate, %	Fresh weight, mg	Nature of callus
2,4-D	BA
0	0	0d	0h	-
	0.5	0d	0h	-
	1	0d	0h	-
	2	0d	0h	-
0.5	0	88 ± 10.95c	94±28.18g	Little callus growth
	0.5	96±8.94ab	125 ± 22.20fg	friable
	1	100a	146.8 ± 23.23ef	compact
	2	92±10.95bc	149.7± 32.06ef	compact
1	0	100a	116.4±26.20fg	Little callus growth
	0.5	100a	180.4 ± 35.68cde	friable
	1	100a	187.3 ±19.03cd	friable
	2	100a	184.3±31.69cd	compact
2	0	88±17.88c	112 ±28.5g	Little callus growth
	0.5	100a	147.2±26.29ef	friable
	1	100a	164.1 ±23.02de	friable
	2	100a	174.78±14.27de	compact
	Kin	-	-	-
0	0.5	0d	0h	-
	1	0d	0h	-
	2	0d	0h	-
0.5	0.5	100a	148.4 ± 13.43ef	friable
	1	100a	174.4±22.35de	friable
	2	100a	164.6± 37.94de	compact
1	0.5	100a	253.2±30.52a	friable
	1	100a	212.4 ±19.57bc	friable
	2	100a	226.18± 22.56ab	compact
2	0.5	100a	173.24±46.23de	friable
	1	100a	187.4±22.12cd	friable
	2	100a	196.8±21.42bcd	compact

The data represent the mean ± standard deviation of three independent experiments. The means followed by the same letters are not significantly different by Duncan’s multiple range test (P < 0.05).

Table 2.

Effect of the exogenous plant growth regulators on callus induction of leaf explants of Plumbago europeae after 28 day.

Auxin, mg/l	Cytokinin, mg/l	Callus induction rate, %	Fresh weight, mg	Nature of callus
2,4-D	BA	-	-	-
0	0	0e	0f	-
	0.5	0e	0f	-
	1	0e	0f	-
	2	0e	0f	-
0.5	0	0e	0f	swelling
	0.5	92 ±10.95ab	68.82 ±19.13e	Little callus growth
	1	92 ±10.95ab	80.24±23.69e	Little callus growth
	2	80±14.14cd	76.02±20.79e	Little callus growth
1	0	100a	91.2±28.8e	Little callus growth
	0.5	100a	130.8± 18.78c	friable
	1	100a	148.66± 38.5bc	friable
	2	84 ±16.73bc	129.4±18.74c	compact
2	0	72±10.95d	95.2±29.91de	Little callus growth
	0.5	84±8.94bc	123.9± 22.046cd	friable
	1	100a	136.1± 23.01bc	friable
	2	96±8.94a	143.5±14.13bc	compact
	Kin	-	-	-
0	0.5	0e	0f	-
	1	0e	0f	-
	2	0e	0f	-
0.5	0.5	96±8.94a	91± 23.025e	Little callus growth
	1	100a	99.04±22.54de	Little callus growth
	2	92±10.95ab	94.8±27.20de	Little callus growth
1	0.5	100a	147.8±20.03bc	friable
	1	100a	179.1±14.45a	friable
	2	100a	165.78±47.05ab	compact
2	0.5	92±17.88ab	132.4±38.58c	compact
	1	96±8.94a	155±15.41abc	friable
	2	100a	166.02± 21.89ab	compact

The data represent the mean ± standard deviation of three independent experiments. The means followed by the same letters are not significantly different by Duncan’s multiple range test (P < 0.05).