Phytochemicals Analysis in Callus, Tissue Cultured and Conventionally Propagated Plant Roots, and Cell Culture of Plumbago indica L

Aims: Plumbago indica L. commonly known as rose-coloured leadwort in English is widely used in the treatment of rheumatism, paralysis, leprosy, headache, leukoderma, enlarged glands, scorpionsting, cancer, ophthalmia, secondary syphilis, dyspepsia, haemorrhage, piles, flatulence and loss of appetite etc. in traditional medicinal systems. Plumbagin is one of the major phytochemicals that is responsible for most of the above biological activities of P. indica. This plant is gradually decreasing from its natural habitats due to over exploitation for medicinal purposes. The use of tissue culture plants for mass scale cultivation and phytochemical extraction can protect P. indica that grows in the natural environment. The development of cell culture allows extracting plumbagin without growing plants in the field. Study Design: Quantitative and qualitative comparison of phytochemicals in tissue cultured and conventionally propagated P. indica plant roots and callus was done using HPLC and GC-MS analysis. Methodology: The amount of plumbagin present in eight months old field-grown tissue cultured and conventionally propagated (shoot cutting) plant roots, callus, and cell culture were quantified by HPLC analysis and phytochemicals were identified qualitatively using GC-MS analysis. Results: Conventionally propagated plant root extract had the highest plumbagin content 23.53 ± 5.34 μg/mL when compared with tissue cultured plant roots (12.90 ± 2.42 μg/mL), callus (0.009 Original Research Article Priyanjani et al.; JPRI, 34(41B): 22-30, 2022; Article no.JPRI.87948 23 μg/mL), cell culture pellet (0.015 ± 0.01 μg/mL) and cell culture supernatant extract (4.23 ± 2.07 μg/mL). The GC-MS analysis revealed that conventionally propagated plant root extract contained a higher number of phytochemicals (16) while tissue cultured plant roots contained only four. Callus extract contained only n-Hexadecanoic acid. Nine phytochemicals were detected in cell culture pellet extract while only three phytochemicals were detected in the supernatant. Conclusion: Tissue cultured P. indica roots can be also used as a source to extract plumbagin in mass scale, yet conditions need to be optimized in order to produce high level of plumbagin. The study confirmed not only the possibility of obtaining plumbagin through cell cultures in a bioreactor system but also products that are mainly secreted into the medium.


INTRODUCTION
Plumbago indica (Plumbaginaceae) is a perennial herb or small shrub that grows well in warm tropical climates. This plant was originated in the Sikkim and Khasi hills of India and migrated to other neighboring countries including Sri Lanka. This plant is cultivated worldwide due to its valuable medicinal root system [1]. This plant contains different phytochemicals that belong to alkaloids, flavonoids, saponins, glycosides, and tannins [2]. Different compound natures of phytochemicals have possessed a wide range of biological activities. Tannins have been found to possess wound healing, antibacterial, anti-inflammatory, hypoglycemic and antidiarrheal activity. Flavonoids are free radical scavengers that protect cells from oxidative cell damagers and show antidiarrheal, anti-inflammatory and hypoglycemic, anticancer, anti-allergic, and antimicrobial activities. Alkaloids are heterocyclic indole compounds that possessed anti-inflammatory, hypoglycemic, hypotensive, anticonvulsant, antiprotozoal, antimicrobial, and antimalarial activities [3].

Preparation of standard curve for Plumbagin
Different concentrations (10.0 -200.0 µg/mL) of standard plumbagin solutions were prepared by diluting 1.0 mg/mL stock solution using ethanol and subjected to HPLC analysis.

Extraction of cell culture
To obtain cell culture extracts, three weeks old calli were cultured on liquid MS medium supplemented with 2.5 mg/L 2,4-D, 1.5 mg/L IBA and 1.0 mg/L NAA. Cell cultures were maintained at 150 rpm on shaker at 25 ± 1 0 C and 16 h photoperiod for three weeks. Phytochemical extraction of cell culture was carried out according to the procedure described by [9]. An aliquot of 50.0 mL was centrifuged at 6,000 rpm for 10 min. The supernatant was extracted thrice with an equal volume (50.0 mL) of ethyl acetate using a separatory funnel at room temperature. Excess ethyl acetate was evaporated using the rotary evaporator at 40 0 C under reduced pressure. Pellet obtained from centrifuged cell culture was crushed with a pinch of acid-washed neutralized sand. Then extracted thrice with 20.0 mL of absolute methanol ((Loba Chemie Pvt. Ltd., Bombay, India)). Excess methanol was evaporated at 40 0 C using the rotary evaporator to obtain crude extract. There were three replicate extractions from each sample. Extracts were weighed and dissolved separately in 2.0 mL of absolute ethanol. All samples were filtered through a 0.45 µm HPLC syringe filter before being subjected to HPLC analysis.

HPLC analysis of samples
HPLC analysis was done according to the procedure described by Muhammad et al., (2009) with few modifications. A volume of 10.0 µL was injected separately into HPLC for analysis. The HPLC was performed by using the C18 column (length 150.0 mm, inner diameter 4.6 mm, and particle size 5.0 µm) (Ultimate 3000, ThermoFisher Scientific, Germany) with a diode-array detector set to a wavelength 254 nm. Separation was carried out at 33 0 C. The mobile phase consists of 90: 10 mixtures of absolute methanol and deionized water at a constant flow rate of 1.25 mL/min. Resulted chromatograms were analyzed to determine the plumbagin content in each.

Preparation of plant crude extract Samples
The same crude extracts used for HPLC analysis were used for GC-MS analysis. Samples for analysis were prepared by sonicating 0.2 g of crude extract (obtained by extracting dried calli, roots of tissue cultured, and conventionally propagated plant) with absolute ethanol for 30 min at 30 0 C separately. Then solutions were concentrated up to 2.0 mL before being filtered through a 0.45 µm syringe filter and subjected to GC-MS analysis. Same cell culture extractions prepared for HPLC analysis were used for GC-MS analysis.

GC-MS analysis
An aliquot of 2.0 µL from each sample was injected separately for analysis. Agilent 7890B Gas Chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with MS-5975C inert mass selective detector (MSD) with triple-axis detector was used for GC-MS analysis. The gas chromatograph (GC) was equipped with HP-5MS 5% phenyl methyl siloxane column (Length -30.0 m, width -250.0 µm, Film thickness -0.25 µm). The oven temperature was initially maintained at 50 0 C for 5 min holding time and raised from 50 to 250 0 C at a rate of 2 0 C/min with zero holding time and the total run time was 105 min. Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1.0 mL/min. The identification of compounds was conducted using the NIST08 library database.

Determination of Plumbagin Content in Callus, Tissue Cultured and Conventionally Propagated Plant Roots and Cell Culture by HPLC Analysis
Ethanol is reported as the most suitable solvent for P. indica root extraction than ethyl acetate, isopropanol, dichloromethane, and diethyl ether. Ethanol has given the highest yield of crude (11.5% w/w) and the highest total plumbagin derivative content (5.79 mg/g) when compared with other solvents. In addition to that, the use of ethanol has another benefit in herbal medications as it has low toxicity. Ethanol extracts are possible to use in herbal preparations [10]. Calli contain rapidly dividing undifferentiated cells. It is a well-known fact that the production of phytochemicals by undifferentiated cells is lower than by differentiated cells. Cell culture supernatant extract contained higher content of plumbagin (4.23 ± 2.07 µg/mL) when compared with cell pellet extract (0.015 ± 0.01 µg/mL). This shows that plumbagin produced are secreted into the medium even without any elicitors in cell culture. HPLC chromatograms for plumbagin (0.4 mg/mL), P. indica tissue cultured and conventionally propagated plant root extracts, cell culture pellet and supernatant extracts and callus extract were shown in Fig. 1.
Plumbagin content in twelve months old tissue cultured field-grown, conventionally field-grown, and hydroponically grown P. indica plants was 1.80 ± 0.25, 1.33 ± 0.15, and 1.08 ± 0.01 g of plumbagin per 100 g of dry raw material, respectively. Dry callus induced from leaf explants on MS medium supplemented with 2.0 mg/L BA and 3.0 mg/L IBA has contained a considerably low amount of plumbagin (0.26 ± 0.02 g per 100 g of dry raw material) [11]. The above literature has proven tissue cultured plants as the best source of plumbagin. However, in contrast, the current study showed higher plumbagin content in conventionally propagated plants Plumbagin is one of the main phytochemicals present in P. indica roots. Production of phytochemicals can be varied according to the nutrient composition of the soil, developmental stage of the plants, climatic season [11] state of mother stock, and other environmental factors. Also, both researchers used two different extraction methods and solvents for phytochemical extractions. In the present study, 10.0 g of dried root sample was extracted with 100.0 mL of absolute ethanol using the Soxhlet apparatus for 4 h. As mentioned in [11], the extraction was carried out by refluxing 2.0 g of dried root sample with methanol for 1 h. The extraction method and solvent used for extraction also can affect the plumbagin content in the tested crude extract.
Many members of Plumbaginaceae family contain plumbagin as one of the active ingredients in the root system. It is reported that P. indica contains a higher amount of plumbagin than P. capensis and P. zeylanica roots. (0.569%, 0.429%, and 0.247% w/w of plumbagin, respectively) [12]. The quantification technique and extraction procedure used by [12] is different than the one used in the present study. Plumbagin was quantified by the HPTLC method and extraction was done by static cold maceration using 50% methanol (hydro alcohol) with 100.0 g of dried roots. The difference in results obtained might have been affected by all the above factors.

GC-MS Analysis of Tissue Cultured and Conventionally Propagated Plant Roots, Callus, and Cell Culture
The GC-MS analysis also revealed that conventionally propagated plant root extract contained the highest number of chemical compounds (16 compounds) than tissue culture plant root extract (4 compounds) and callus extract (1 compound). Cell culture pellet extract only contained nine phytochemicals while supernatant extract contained only three phytochemicals having more than 70% similarity. Similarity percentages of phytochemicals present in different samples are shown in Table 2.

CONCLUSION
The conventionally propagated plant root extract has a higher number of phytochemical compounds and higher plumbagin content when compared with tissue cultured plant root extract, calli, cell culture supernatant, and pellet extracts. Even though, conventionally propagated plant roots are the best source to extract plumbagin, tissue cultured plant roots also have a considerably high amount of plumbagin. Thus, results revealed that tissue cultured P. indica roots can be also used as a source to extract plumbagin on a mass scale, yet conditions need to be optimized to produce a high level of plumbagin. The study confirmed not only the possibility of obtaining plumbagin through cell cultures in a bioreactor system but also products are mainly secreted into the medium. Therefore, it is not necessary to use mechanical product secretion. Production of plumbagin in cell culture should be further increased using elicitors or manipulating other environmental factors before using this as a commercial scale plumbagin production method.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by research grant of University of Sri Jayewardenepura.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.