INTRODUCTION:

Shatavarin IV, one of the steroidal saponin is the major bioactive phytoconstituent from the root of Asparagus racemosus having anticancer activity¹. There are 22 species of Asparagus found in India and Asparagus racemosus is the most frequently used herb in Ayurvedic Rasayana due to its wide range of pharmaceutical and medicinal uses². The medicinal value of the plant is due to the major bioactive compound produced within plant body. The therapeutic values of Asparagus racemosus is mainly due to the presence of Shatavarin IV.

The root extract of A. racemosus is known to be used in dyspepsia, nervous disorders, diarrhoea, dysentery, inflammations, tumors, hyperdipsia, hepatopathy, neuropathy, cough, hyperacidity, bronchitis and certain infectious diseases³. The high medicinal value leads to overexploitation of the primary source of Shatavarin IV. The declining condition of the wild population of Asparagus racemosus in India might be due to the over collection and exploitation. In India, A. racemosus is placed in the nearly threatened category in the states of Chhattisgarh and Madhya Pradesh and even endangered in West Bengal⁴. This led to the searching of other naturals sources of Shatavarin IV to reduce the collection pressure on the primary source and thereby conserving the wild population. The possible natural sources of Shatavarin IV might be other species of Asparagus genus for alternative commercial production and industrial applications. Limited species of Asparagus genus which have been screened for Shatavarin IV excludes the species like Asparagus densiflorus, Asparagus setaceus, Asparagus plumosus and Asparagus sprengeri, were taken for the investigation. The wild verity Asparagus racemosus was also included in the study for comparative analysis. Asparagus densiflorus is an environmental management plant material which is effective in treating industrial effluent and soil remediation process⁵. It is also known to exhibit anticancer activity⁶. Asparagus setaceous is known for its high medicinal and commercial value but scientific research is limited for this species⁷. Asparagus plumosus is an important commercial greenhouse plant in Denmark⁸. Asparagus sprengeri is an ornamental plant and less explored for their phytochemical analysis⁹. This plant is known to exbibit molluscicidal properties against Biomphalaria alexandrina snails¹⁰. The plant is used as herbal medicine in Iraq and the essential oil extracted from this plant showed antibacterial activity¹¹. Moreover, the literature regarding the selected species of Asparagus genus shown that these plant species were largely used for ornamentation which were of great economic values. The phytochemical study is very less in these plants according to the previous reports. However, the species of Asparagus have been used as immunostimulant, antibacterial, reproductive agents, antioxidant, anti-inflammatory and antihepatotoxic⁶. In Ayurveda, A. racemosus root is being used to cure a spectrum of diseases like diarrhoea, nervous disorders, dysentery, dyspepsia, tumours, inflammations, neuropathy, hyperdipsia, hepatopathy, cough, hyperacidity, bronchitis and certain infectious diseases¹².

Therefore, the present investigation is carried out for a novel cause to determine the presence of Shatavarin IV which would give some idea about the phytochemical profile of these plants. The presence of such compound would increase the medicinal importance and commercial cultivation of these less explored species of Asparagus genus. They could serve dual purposes of ornamental and medicinal use. Also, the identification of Shatavarin IV would reduce the collection pressure on the primary source, which in turn would conserve the wild population of Asparagus racemosus. The aim of this study is also designed for the development of HPTLC fingerprint of these selected plants for proper identification and quality assurance, thus having significance for commercial use and future research.

MATERIALS AND METHODS:

Plant sample:

The plants of five Asparagus species were procured from a local nursery and identified by taxonomist, Prof. PC Panda, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha. These were grown in an experimental green house for three generations. The fresh roots of mature plants were collected in a harvesting month of Asparagus during September-October. The root samples of five species of Asparagus were shade dried and the coarse powder (60 mesh) was obtained by pulverization in a mechanical grinder. The powdered samples were stored for HPTLC analysis.

Chemicals and reagents:

The standard chemical of Shatavarin IV (90% purity) was procured from Natural Remedies Private Limited. Ethyl acetate (99.7%), Methanol (99.7%), Sulfuric acid (98%) and Water (HPLC grade) were obtained from Merk Life Science Private Limited, Mumbai, India. p-Anisaldehyde (98%) was purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Acetone and Acetic acid glacial (100%) were obtained from Merck Specialities Private Limited, Mumbai, India. The HPTLC silica gel 60 F₂₅₄(10×20cm) plates were procured from Merck KGaA, Darmstadt, Germany.

Extraction procedure for Shatavarin IV and Standard Solution preparation:

The powdered plant samples were extracted using methanol. 0.8g of powdered root sample was added to 10ml of methanol and placed in Water bath (MS Stirring Water Bath) at 60^oC for 1 hours. It was then filtered through syringe filter of 0.22µm pore size. The filtered solution was used in HPTLC analysis. The standard chemical of Shatavarin IV was prepared by dissolving in methanol.

HPTLC Chromatographic Conditions:

The chromatographic analysis was done by spotting the extracted solution of roots of A. racemosus and standard Shatavarin IV on pre-coated silica gel 60 F₂₅₄ (10×20 cm) HPTLC plates as stationary phase. The samples and standards were applied to the plates with the help of CAMAG Linomat V sample applicator and a 100µL syringe. The band length of application was 8mm and application rate was 28-34 kPa (4-5 lbf/in²) in nitrogen aspirator for application. The separation between the bands was 12mm. The linear ascending plate development was achieved through the mobile phase of ratio of ethyl acetate-methanol-water (7.5:1.5:1, v/v/v) using 20cm × 10cm twin-trough glass chamber (CAMAG) saturated for 20 minutes in mobile phase at normal room temperature. The volume of mobile phase was 20mL (10mL in each trough) and the run distance of mobile phase on the plate was set to 90mm. The developed plate was then air dried using an air-dryer. The plates were visualized in a CAMAG UV cabinet at 254nm and 366nm light. A simple glass derivatizer was used for derivatization where the plate was dipped in anisaldehyde‒sulfuric acid reagent with the help of a derivatizer and subsequently heating the plate at 110°C in hot-air oven for 5 min. The plates were then viewed at 366 nm wavelength in CAMAG UV cabinet and the photos were taken both in 366nm wavelength light and the fluorescence light after derivatization. The plate was scanned using CAMAG TLC scanner 4 equipped with winCATS Software (Slit dimension 6.00 × 0.45). The scanning was done at 425nm and the amount of Shatavarin IV present in the samples was quantified. The spectra matching was carried out in sample and standard by taking the absorbance of Shatavarin IV between 300-700nm.

Method Validation:

The method validation of quantitative analysis was done in terms of linearity, limits of detection (LOD) and quantiﬁcation (LOQ), precision (Intra-day and Inter-day) and recovery test according to the guidelines set by International Conference on Harmonization (ICH)¹³. The calibration curve was done by taking six concentrations of Shatavarin IV for the quantification Shatavarin IV in the plant samples. The curve was done by taking Shatavarin IV in ng/band (X-axis) verses Area Unit (Y-axis) of the band.The limit of detection (LOD) is the lowest concentration of an analyte at which the peak area of the signal is at least three times greater than signal to noise ratio (S/N ≥ 3). The limit of quantification (LOQ) is the minimum concentration of an analyte at which the peak area is at least ten times greater than the signal by noise ratio (S/N ≥ 10). The precision was determined in terms of Intra-day, injecting the replicate solution of standard for three times (0, 3, 6hrs) within a day and Inter-day, injecting the same solution of standard for three consecutive days (0, 24, 48hrs). The recovery test was done by the standard addition method, where a known amount of standard was added to the analyzed sample and then reanalyzing.

RESULTS AND DISCUSSION:

Optimization of extraction condition and mobile phase:

The most important step in the phytochemical analysis is the extraction of desired bioactive compound. This is based on the principle to elute maximum desired bioactive compound from the complex plant matrix without destroying the chemical nature of the same¹⁴. Here, different combination of solvents (methanol, acetone and water), temperature (30°C, 40°C, 50°C and 60°C) and time of extraction (20, 40, 60 and 80 minutes) were analysed and the best combination to elute maximum Shatavarin IV was found to be methanol in 60°C for 60 minutes. Although, alcohols are commonly used for the extraction of saponins but methanol is widely used alcohol for better extraction yield¹⁵. According to literature, methanol is the most effective solvent to extract more of saponins from the plant matrix compared to other common solvents¹⁶. Previously, methanol was used to extract saponins from Momordica cochinchinensis Spreng.¹⁵ and Salicornia herbacea¹⁷. The saponin extraction from the Asparagus species were also done using methanol as extraction solvent for chromatographic analysis^18-19. The major bioactive compound Shatavarin IV was determined by the methanolic extracts of Asparagus racemosus by a number of researchers^20-22. Therefore, the use of methanol in appropriate temperature and duration for the quantitative determination of Shatavarin IV is justified. The temperature parameter of the phytochemical extraction plays an important role in extracting the desired bioactive compound and chemical stability of the compound. In our study, by increasing the extraction temperature upto 60°C increases the extraction yield but gradual small increase in temperature beyond that the yield was declined and solvents started to evaporate (after ⁓65°C). The surface tension and viscosity of the extraction solvent decreases at high temperature and at this condition the solvent can weaken the cell wall and cell membrane of the plant cell in order to elute maximum desired phytochemicals²³. The decrease in yield of desired compound in our study might be due to degradation of phytoconstituents at higher temperature. In the above condition of temperature and type of solvent, the yield increased upto extraction duration 60 minutes and beyond that value decline. The declining of yield due to longer exposure to higher temperature might due to degradation of phytochemicals in the plant cells²⁴. The mobile phase (ethyl acetate-methanol-water, 7.5:1.5:1, v/v/v) used in our study efficiently separate the bands on the chromatographic plates. The mobile phase gave bands of good resolution with stable peaks. The mobile phase used in our study was standardized by earlier researchers for better separation of bands in chromatographic determination of Shatavarin IV^22,25. The twin-trough glass chamber was saturated with chromatographic paper and mobile phase for 20 minutes in order to get uniform distribution of mobile phase.

Calibration curve and method validation:

The calibration curve for quantitative analysis of Shatavarin IV has shown good linearity and appropriate coefficient of correlation (R²) of 0.9968. The linear range of the Shatavarin IV in the calibration curve was between 72-432ng/band.The quantitative analysis was done by using a regression equation i.e., y=14.25x+779.45. The limit of detection (LOD) and limit of quantification (LOQ) was determined using signal-to-noise ratio of the chromatographic peak analysis. The LOD of Shatavarin IV was found to be 24ng and LOQ of Shatavarin IV was found to be 72ng. Prior to HPTLC analysis of the samples, the method was validated for proper identification and quantification of the target constituent. The outcomes of precision tests were satisfactory according to the standard requirement of quality control criteria set by ICH guidelines¹³. The relative standard deviation (RSD) of intra-day and inter-day precision was found to be 1.63% and 1.69% respectively which were the acceptable range of validation tests (Table 1). The recovery percentage of Shatavarin IV was found to be more than 97% with less than 0.97% RSD. The results of the method validation show the robustness of the analytical methods used in our study.

Table 1. Precision of Shatavarin IV by HPTLC method.

Component	Precision	Amount applied (ng)	Amount found (ng)	Mean	RSD (%)
Shatavarin IV	Intra-day	705	703	701	1.63
			699
			701
	Inter-day	705	703	702.33	1.69
			700
			704

RSD: Relative standard deviation, ng: nanogram

Identification and quantification of Shatavarin IV:

The Shatavarin IV was identified in the samples by comparing with the retardation factor (R_f) and matching the UV spectra of standard Shatavarin IV. The R_f was found to be matched at 0.55±0.05 and the maximum absorbance was obtained at 425nm (Figure1). The colour visualisation of the bands and band matching was also used as an identification criterion. Almost all the plant showed the presence of Shatavarin IV in them. The identified bands were quantified by using the calibration curve and were expressed in percentage. The amount of Shatavarin IV was found to be highest in Asparagus racemosus (0.22%) among all the five species (Table 4). The lowest or negligible amount of Shatavarin IV was reported from Asparagus densiflorus (0.01%). The amount of shatavarin IV were found to be 0.08%, 0.04% and 0.06% in A. setaceus, A. plumosus and A. sprengeri respectively. Growing the plants for three generations under homogeneous environmental conditions collaborate that the variation in their chemical profiles is genetically determined and are not due to environmental influences²⁶. After Asparagus racemosus, Asparagus setaceous had the highest content of Shatavarin IV of 0.08%. Apart from genetic variations, morphology of the plants plays important role in yield and production²⁷. There was a great variation in the morphology of the roots of five species of Asparagus genus, which might contribute for the variation in the production and accumulation of secondary metabolites (Figure 2) which needs further validation. Shatavarin IV was also reported from another species of Asparagus i.e., Asparagus gonoclados. Previously, the alcoholic and aqueous extracts of Asparagus gonoclados was screened qualitatively for the presence of Shatavarin IV^28-30. However, the quantitative and qualitative analysis of Shatavarin IV in four species of Asparagus genus has been reported for the first time in the current research work.

Table 2. Quantitative estimation of Shatavarin IV (%) in five different species of the genus Asparagus.

Serial number	Name of the species	Shatavarin IV (%)	Voucher number
1	Asparagus racemosus	0.22	1965/CBT
2	Asparagus densiflorus	0.01	1966/CBT
3	Asparagus setaceus	0.08	1967/CBT
4	Asparagus plumosus	0.04	1968/CBT
5	Asparagussprengeri	0.06	1969/CBT

HPTLC fingerprint:

There are number of chromatographic fingerprinting techniques useful to ascertain identity and composition for identification and quality control of raw material and finished herbal product. The most frequent among them are high-performance liquid chromatography (HPLC), gas chromatography (GC), High-performance thin layer chromatography (HPTLC) and mass spectrometry (MS). The HPTLC fingerprinting method is the most potent tool because of its simplicity and reliability^30-31. Previously, many of the important medicinal plants has been analysed for their bioactive compounds though HPTLC analysis^32-37. The HPTLC fingerprint has been used for phytochemical profiling and comparative assessment^38-39. The HPTLC method used in a particular study should be validated^40-41and, in our study, we did the same. The developed HPTLC fingerprint of five species of Asparagus genus would serve as reference for future study (Figure 3). It could be used for identification and quality control purposes of these plants in future. The fingerprint has been developed using methanolic plant root extract with p-anisaldehyde as derivatisation reagent.

Figure 3. HPTLC Fingerprint of five species of Asparagus genus. Lane S-Standard (Shatavarin IV), A- HPTLC Fingerprint at 254nm wavelength of light (before derivatization), B- HPTLC Fingerprint at 366nm wavelength light (after derivatization), C- HPTLC Fingerprint at normal florescent light (after derivatization). Lane 1-2 (Asparagus racemosus), Lane 3-4 (Asparagus densiflorus), Lane 5-6 (Asparagus setaceous), Lane 7-8 (Asparagus plumosus) and Lane 9-10 (Asparagus sprengeri)

CONCLUSION:

The present investigation revealed the identification and quantification of Shatavarin IV in four species of Asparagus genus for the first time. Also, the HPTLC fingerprint was developed for five species of Asparagus for the first time.It could be used as reference for future research on these species and their quality control. The identification of Shatavarin IV in other species of Asparagus would explore new source of it and would be helpful in compensating the high demand of Shatavarin IV. This would reduce the collection pressure on the wild population of primary source of Shatavarin IV i.e., Asparagus racemosus. The study revealed that highest amount of Shatavarin IV is quantified in Asparagus setaceous after Asparagus racemosus. So, Asparagus setaceous would be the potential source of Shatavarin IV after A. racemosus and other three precedes the list.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to Prof. (Dr.) S.C. Si, Dean, Center of Biotechnology and Prof. (Dr.) M.R. Nayak, President, Siksha ‘O’ Anusandhan University for their support and encouragement. Moreover, the authors would like to thank the Science and Engineering Research Board, Govt. of India for their extramural research grant (Grant No. EMR/2016/001802).

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Received on 09.02.2022 Modified on 14.06.2022

Research J. Pharm. and Tech 2023; 16(6):2615-2621.

DOI: 10.52711/0974-360X.2023.00429