Evaluation of Seasonal Variation and Procurement Time for
Eclipta prostrata (L.) L.C.

 

Cheemalapati Venkata Narasimhaji¹*, A K Mangal², Rekha Prabu¹, Ilavrasan R¹,
Narayanam Srikanth²

¹Captain Srinivasa Murthy Central Ayurveda Research Institute (CSMCARI), CCRAS, Chennai.

²Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi.

 

ABSTRACT:

Efficacy of the medicinal plants depends on the nature of primary and secondary metabolites present and assay of metabolites in raw botanical parts used for the drugs. Quantity and nature of metabolites that are presented are correlated with the growth of the plants, herbs and the season in which they are collected. Hence, it is important for a search of the best time and season for collection, based on the part used of medicinal plants, the assay of the secondary metabolites present and other morphological features for assess the maturity etc., In this study, it is aimed to evaluate the seasonal variation in terms of assay of wedelolactone through RP-HPLC and validate the best procurement time for the whole plant of Eclipta prostrata L. besides botanical verification and pharmacognostic authentication. The developed method was validated, results of analytical findings were summarized by all three triplicate tests for the three different solvent extracts of whole plant material of Bhringraj for all collected six seasons and found more abundance of wedelolactone in the range of 0.146-0.209 % w/w in Sharad ritu (Sep-Oct)/ Autumn season.

 

 

INTRODUCTION:

Eclipta prostrata L.   (syn. Eclipta alba (L.) Hassk.) (Bhringraj) is a wild grown an annual herb, popular for ethno medicinal importance and belongs to the Asteraceae family. In India E. prostrata is commonly known as Bhringraj and as false daisy in English. It is a famous traditional medicine in the three major forms of traditional medicinal systems namely Ayurveda, Unani & Siddha in Indian subcontinent, China, many other countries in the world and is also edible natural product. The Ayurvedic Pharmacopoeia of India   considers the plant as hepatoprotective¹.

 

E. prostrata L. is used as a tonic, de-obstruent in hepatic and spleen enlargements and skin diseases. The plant juice is administered for jaundice. The leaf juice with honey is a trendy remedy for catarrh in infants.

The juice of the leaves boiled with coconut or sesame oil is used for applying the head to provide the abundant black hair. The fresh plant is considered anodyne and absorbent. The plant is used as a dyeing herb in tattooing². The herb is found throughout the year in marshy places.

 

In ayurvedic treatises various important indications are mentioned. In Charaka and Sushrut samhita it is indicated to use for cough and dyspnoea^3,4. Vagbhata recommends its use as a tonic and in vitiligo⁵. Charaka and Chakradatta mentioned its role in blackening of hair^6,7. It is widely used to treat respiratory diseases such as diphtheria, pertussis and tuberculosis in the traditional medicine of China^8,9, it exhibits hepatoprotective^10,11, anti-tumor¹² and other biological activities^13,14. In Brazil, its extracts are used to treat asthma^15,16,17. It has been reported that the methanol extract of this plant appreciably reduce the effect of pulmonary fibrosis in mice¹⁸. Wedelolactone, a main component of E. prostrata, protects human bronchial epithelial cell injury¹⁹ and fibrosis process of activated hepatic stellate cells²⁰. It was reported for folklore medicine and for many traditional uses such as for black hair, body tingling effect, ringworm infection, fertility agent, diarrhea, for toes pains, head ache and jaundice²¹. It was also reported for anthelmintic activity^22,23 and immunomodulatory effect²⁴ and ameliorative potential²⁵.

 

Phytochemical evaluation of extracts of stem of E. prostrata²⁶ and it was reported for a number of bioactive chemicals besides wedelolactone such as stigmasterol, stigmasterol-3-O-glucoside, daucosterol, demethylwedelolactone, 𝛼-amyrin, oleanolic acid, ursolic acid, luteolin, luteolin-7-glucoside, apigenin, protocatechuic acid, 4-hydroxy benzoic acid, heptacosanol and ecliptalbine etc^27,28,29,30. In recent years, many pharmacological studies have observed for wedelolactone for a wide range of biological activities, including hepatoprotective, antiosteoporosis, enzyme inhibition, antiphlogistic, anti-inflammatory, promoting cell proliferation, and other effects. Wedelolactone has been found to be useful in the treatment of osteoporosis, pulmonary embolism, and ulcerative colitis³¹.

 

Efficacy of the medicinal plant materials depend on the nature of primary; secondary metabolites present in it and its degree of abundance. Medicinal properties play directly or indirectly dependant on the concentrations of secondary metabolites of that plant for its superiority or in terms of quality. The total amount of secondary metabolites interrelated with the growth of the herbs and plants, and certainly depends on the collection of the plant materials and its parts in which season they are acquire. Hence it is important to find out the best season for the collection of raw botanical materials, its useful parts for therapeutic efficacy. In this study line, it was carried out the seasonal variation of some important medicinal plants namely Justicia adhatoda L., Withania somnifera (L.) Dunal, Terminalia arjuna, Andrographis paniculata for their secondary metabolites vasicine, wthanolide A, ellagic acid and andrographolide respectively^32,33,34,35. Now, in the present study, it is aimed to evaluate the best procurement time and seasonal variation for the whole plant of E. prostrata with the assay of wedelolactone, besides pharmacognostic authentication.

 

Wedelolactone

 

MATERIALS AND METHODS:

Collection of Plant material and authentication:

Whole plant of Bhringraj (The habitat, A. No. 23596), seasonal wise Shishir ritu/winter (Jan-Feb), Vasant ritu/spring (Mar-Apr), Grishma ritu/ summer (May-Jun), Varsha ritu/ monsoon (Jul-Aug), Sharad ritu/ autumn (Sep-Oct), Hemant ritu/pre-winter (Nov to Dec) were collected from Regional Ayurveda Research Institute, Jhansi-garden and authenticated by the botanist of RARI, Jhansi. The collected plant materials were washed under running fresh tap water to remove adherent soil and dirt. The whole plant samples were collected in triplicate was preserved in 70% alcohol for histological studies. The collected and washed plant materials were subjected to shade drying for about four weeks, were further crushed to powder and passed through the mesh 22 and stored in air tight container for further analysis.

 

Morphology, Macroscopic and Microscopic analysis:

Collected plant materials were observed carefully for its identical morphological characters such as colour, shape and size etc. The measurements were taken by scale through naked eye and photographs were documented with Nikon DSLR Camera. Comparison of macroscopic characters for each different season was recorded with the help of floras⁷. Microscopic examinations of different parts of plant were observed according to the standard method^8-10. Transverse sections of plant parts were prepared (Fig.2), stained with saffranin and fast green as per procedure. Same procedure was followed for powder microscopy (Fig.3). The photographs of microscopic features for plant materials were documented by bright field microscope with digital camera.

 

Test Solution and Extraction of Plant Material:

Each 5 g of dried, powdered whole plant material collected in all six seasons was extracted with 200 ml of methanol, ethanol and hydro-alcohol (95%) separately by using soxhlet for 24 h. The extracts were evaporated under reduced pressure get dry residues (Table-1). The residues were weighed in triplicate and dissolved in methanol to make up the volume of a 10 ml volumetric flask to obtain the test solutions.

 

Standard Solution:

2.2 mg of wedelolactone was accurately weighed, dissolved in HPLC grade methanol and made up the volume in a 10 ml volumetric flask to obtain 0.22 mg/ml.

 

Chemicals and Reference Standards:

Standard marker compound wedelolactone was purchased from Natural remedies Pvt. LTD. Ethanol, methanol, acetonitrile and water (All HPLC grade) was purchased from Merck.

 

Instrumentation and HPLC Analysis:

HPLC analysis was performed on Agilent Technologies 1200 series, it consists of 1100/1200 Quaternary Pump, Manual injector, 1200 Variable Wavelength Detector. Separation was achieved on Eclipse XBD C₁₈ –column (dimensions 4.6 mm x 150 mm). Chromatograms were recorded by using Agilent chemstation software. The mobile phase was consisted of methanol and water (70: 30) pumped at a flow rate of 2.0 ml/min. The detection was carried out at 352 nm. An injection volume of both standard and sample was 10μl. The temperature in laboratory was at 25 ± 2°C. All the solutions used for the analysis were filtered through 0.45 μm membrane filter and extracts were filtered through 0.22 μm membrane filter using Thermo fisher and Millipore filtration unit.

 

Calibration and Estimation of Wedelolactone:

The above standard solution was appropriately diluted further to get a concentration of 0.022, 0.044, 0.066, 0.088 and 0.11 mg/ml of standard. Each of the standard solution was run through the HPLC and recorded the respective peak areas. Calibration curve was established for peak area vs concentration of standard applied (Fig-5). 10 μl of each test solution was obtained from all the six seasons of E. prostrata were given in the Table-1 to Table-2 was injected to HPLC, the amount of wedelolactone was estimated in the residues extracted in various test samples from the area   of test solution corresponding to that of standard from the above calibration curve.

 

Method validation:

Specificity was observed with the reagent blank for methanol and water, which were used for preparation of sample and standard. Linearity was established with different concentrations of working standards as described in calibration curve.

Recovery Study:

Recovery studies were carried out by mixing known quantity of standard with the pre analyzed sample. The concentration of these solutions were observed and amount recovered was calculated using formula of % Recovery = 100 × Observed value/True value (Table-3);

Precision (Repeatability):

Precision of analytical method is usually expressed as the standard deviation or Relative standard deviation. It was analyzed by repetition of spiked samples at minimum of five times at varying concentration from 0.022 to 0.11% for analyte of interest in same day, same person and in same conditions. Finally the %RSD of the samples was calculated. (Table-3);

Reproducibility:

Repetition of sample of spiked with lower concentration for five times of analyte in different day, different person and different conditions were analyzed to calculate the % RSD. (Table-3);

 

Ruggedness:

Reproducibility of results obtained for sample by the different analyst, different days and different conditions were recorded. (Table-3)

 

RESULTS AND DISCUSSION:

The whole plant of Bhringraj was authenticated in all six seasons with the macroscopic, microscopic features. Powder analysis was carried out throughout the year for the collected raw botanical materials of each season from the same habitat (RARI garden) (Fig. 1-3).

Root: In macroscopic features observed for root cylindrical tapering at the distal end, 0.5 to 0.8 cm in diameter, bears numerous rootlets, surface faintly striated, fracture fibrous; color creamish brown, taste slightly bitter and odour not characteristic.

Stem: cylindrical, 0.4 to 0.8 cm in diameter, with distinct nodes, internodes being 4 to 7 cm in length; surface pubescent and striated when young, rough when old; fracture fibrous; colour greenish brown; taste slightly bitter; odour not characteristic.

Leaf: opposite, decussate, oblong-lanceolate, 2.5 to 8 cm in length and 1.0 to 2.5 cm in width; brittle, entire or dentate, acute, sub-sessile, pinnate, veins obscure on upper surface, prominent on lower surface, pubescent when fresh, trichomes get detached on drying; colour green, lower surface paler; taste slightly astringent; odour characteristic.

Flower: head, 6 to 8 mm in diameter, solitary or 2 together on unequal axillary peduncles; involucral bracts about 8, ovate to obtuse with appressed white hair; ray flowers white, ligulate, ligule small spreading rarely as long as the bracts, not toothed; disc flowers tubular, corolla often 4 toothed, pappus zero except occasionally with very minute teeth on the top of the achene. Comparison of morphological characters for the all available six seasons were given in Fig.1(a-d). The Bhringraj was observed with white colored flowers in four seasons of Grishma ritu, Varsha ritu, Sharad ritu and Hemant ritu; and otherwise in other two seasons of Shishir ritu and Vasant ritu samples with white flowers and fruiting was observed.

 

In microscopic features, the significance difference was observed the presence of fragments of papillose stigma, outer epidermis of disc-floret, coloring matter in microscopy of sample collected in Sharad ritu (Autumn) only, when compared to other seasons. It was also observed that the pitted xylem vessels in both samples of Varsha ritu (Monsoon) and Sharad ritu (Autumn) only and in all other season samples are not pitted (Fig-2). In powder drug features, the powder is of dark green powder, showed simple, covering, uniseriate, three-celled, short or very long trichomes with all three cells being different from each other as the terminal cell is conical, very small in size with pointed apex, the basal cell is broad and thick-walled, while the one lying in between the two is the longest, warty and with tubercle margin; smaller 3-celled trichomes with smooth margin from disc-floret; upper and lower epidermis in surface view, cells of the former being bigger in size and with straight anticlinal walls, unlike the latter which are with wavy anticlinal walls, embedded with more numbers of anomocytic and anisocytic stomata; small spherical warty pollen grains scattered as such or embedded in the fragment of disc- florets; fragments of fibrous layer of anther showing characteristic thickening and beading of the walls in surface and cross-sectional view, thickening of the side wall of the cells appear as closely packed longitudinal bands; fragments of style and stigma, style composed of thin-walled elongated cells, the epidermal cells of the apices of the stigmas are extended to form rounded or oblong papillae; outer epidermis of disc-floret composed of thin-walled longitudinally elongated cells, which are usually markedly sinuous in outline and embedded with pollen grains, and bear 3-celled small trichomes; fragments of thick-walled polygonal suberised cork in surface view; fragments of longitudinally cut bordered pitted and spiral vessels and fibres from stem and root, and annular vessels from leaf; coloring matter from petiole and stem; reticulated endocarp of achene in surface view; prismatic crystals of calcium oxalate scattered as such or embedded in the cortical cells of stem and mesophyll of leaf (Fig. 3).

 

The marker compound wedelolactone was selected to quantify in each season through HPLC, to evaluate the seasonal variation and to find out the best procurement time of the Bhringraj. For this, all collected seasons plant materials were extracted as mentioned in materials and method section, extractive values found in methanol are more compare to other two solvents of ethanol, 95% hydro-alcohol and residue/extractive values (Table-1). The detection was carried out at 352 nm in a solvent system of methanol and water (70: 30), at a flow rate of 2.0 ml/min with 1.113 retention time (r.t.) and linearity of correlation coefficient (r²) = 0.9999 was established from above working standards. The area 280.144, 587.896, 906.312, 1226.82 and 1549.44 respectively with increased concentrations was observed and with slope value 14443.Tthe limit of detection 0.0047 mg/ml and limit of quantification 0.0145 mg/ml were found from the established linear curve (Fig. 5).The method validation parameters such as recovery study, repeatability, reproducibility, ruggedness etc.,^36,37,38,39 as described in methods section were briefed in a table-3 and findings were summarized in table-4. The residues extracted were injected as a test solution in triplicate of each sample for the estimation of wedelolactone in the validated method. The amount of wedelolactone found low in varsha ritu (0.0373%), more in Sharad ritu (0.1761 % w/w) and in the range of 0.1464 to 0.2090 %, out of all the available six seasons of Bhringaraj given in the Table-2. It was observed the variation in the amount of marker compound from low to high (0.021-0.209% w/w) in all seasons and in methanol extract contains more that is about 0.2090 % when compared to other extracts. Based on above observations and the findings in terms of assay of wedlolactone, the best collection time for E. prostrata is Sharad ritu (Sep-Oct), the autumn season.

 

Fig. 1: (a) E. prostrata flowering plant in Shishir, Vasant and Hemant ritu.(b) Flowering and fruiting in Varsha and Sharad ritu.

(c) Habitat Grishm ritu (flowering). (d) E. prostrata dried drug in Varsha ritu.

 

Table-1: Extractive values for 5 g of each raw botanical sample

 

 

Table-2: Assay of Wedelolactone (in mean value & range)

*Mean: Triplicates of three different variety solvents extracts of E.prostrata plant material (9 test samples).

 

Fig. 2: (a) Ts of E. prostrata stem, 4x: e, epidermis; hyp, hypodermis; cav, cavity; ct, cortex; end, endodermis; xy, xylem; perf, pericyclic fibre; ph, phloem; pi, pith; pcr, prismatic crystals of calcium (b)Ts of E. prostrata stem, 10x: e epidermis; hyp, hypodermis; cav, cavity; ct, cortex; end, endodermis; xy, xylem; perf, pericyclic fibre; ph, phloem; pi, pith; pcr, prismatic crystals of calcium oxalate (c) Ts of E. prostrata leaf, 4x: ue, upper epidermis; pal, palisade; spme, spongy mesophyl; bv, vascular bundle; t, trichome; le, lowwr epidermis; xyv, xylem vessel; ph, phloem; col, collenchymas (d) Ts of E. prostrata leaf, 10x ue, upper epidermis; pal, palisade; spme, spongy mesophyl; bv, vascular bundle; t, trichome; le, lowwr epidermis; xyv, xylem vessel; ph, phloem; col, collenchymas.

  

Table-3: Validation study

 

 

Fig. 3: Powder microscopy E. prostrata whole plant, (a), lower epidermis of leaf showing anisocytic and anomocytic stomata; (b), cork of root in surface view; (c), upper epidermis of leaf; (d), fragments of longitudinally cut xylem vessels, annular and spiral; (e), pollen grains; (f), prisms of calcium oxalate; (g), fibrous layer of anthers in surface view; (h), fibres; (i), trichomes (simple covering uniseriate three celled).

 

Fig. 4: HPLC Chromatogram of E. prostrate  (A).Test solution; (B). Wedelolactone standard

 

 

Fig.5: Calibration curve of  Wedelolactone.

 

Table-4: Validation Parameters

 

CONCLUSION:

The collected plant material was authenticated macroscopic and microscopically; the significance difference was observed, that the presence of fragments of papillose stigma, outer epidermis of disc-floret, coloring matter in the microscopy of sample collected in Sharad ritu (Autumn) only, when compared to other seasons. It was also observed that the pitted xylem vessels in both samples of Varsha ritu (Monsoon) and Sharad ritu (Autumn) and in all other season samples are not pitted. The developed method was validated and all the parameters of validation summarized in the table-4. All collected six seasons of whole plant material of Bhringraj was extracted in three different solvents were analyzed in triplicate and evaluated the seasonal variation in the quantity of marker compound. The results of analytical findings indicates that the amount of wedelolactone found low in varsha ritu (0.0373%), more quantity in Sharad ritu (0.1761 % w/w) and in range of 0.1464 to 0.2090 % w/w in Sharad ritu. In conclusion, based on above observations and the findings in terms of assay of wedlolactone, the autumn season/Sharad ritu is recommended for the best collection time, that is, September to October for E. prostrata.

 

ACKNOWLEDGEMENTS:

Authors are expressing their gratitude to the Director General, CCRAS for financial support. Thanks to Dr. Ravindra Singh, Assistant director (Chemistry) and Dr. Pramila Pant Ex. Assistant director (Chemistry), CCRAS, New Delhi for encouraging to conduct the study.

 

CONFLICT OF INTEREST:

The authors declare No conflict of interest.

 

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Accepted on 21.12.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):4967-4973.