|Year : 2023 | Volume
| Issue : 2 | Page : 134-142
Seasonal assessment of asiaticoside content in Centella asiatica (L.) Urb. by HPLC and pharmacognostical studies to establish its best procurement time
Ch Venkata Narasimhaji1, Anupam K Mangal2, P Rekha3, R Ilavarasan4, Narayanam Srikanth5
1 Department of Chemistry, CCRAS-Captain Srinivas Murthy Central Ayurveda Research Institute, Chennai, India
2 Department of Pharmacognosy, CCRAS-Central Ayurveda Research Institute, Kolkata, India
3 Department of Chemistry, CCRAS-Captain Srinivasa Murthy Central Ayurveda Research Institute, Chennai, India
4 Department of Pharmacology, CCRAS-Captain Srinivasa Murthy Central Ayurveda Research Institute, Chennai, India
5 Central Council of Research in Ayurvedic Sciences, New Delhi, India
|Date of Submission||21-Dec-2021|
|Date of Acceptance||15-Dec-2022|
|Date of Web Publication||31-Mar-2023|
Dr. Ch Venkata Narasimhaji
Department of Chemistry, CCRAS-Captain Srinivasa Murthy Central Ayurveda Research Institute (CSMCARI), M/o AYUSH, Govt. of India, A. Anna Hospital Campus, Arumbakkam, Chennai-106
Source of Support: None, Conflict of Interest: None
BACKGROUND: This study was designed to validate the collection time for the whole plant of Mandookparni [Centella asiatica (L.) Urb.] by assessing the variation in marker compound asiaticoside, a bioactive secondary metabolite with quantitative high performance liquid chromatography in all available seasons along with the observation of morphological and microscopic studies. METHODS: Centella asiatica (L.) Urb. the whole plant was collected from the same habitat in all six seasons described in Ayurveda that is Shishir ritu (Jan–Feb), Vasant ritu (Mar-Apr), Grishm ritu (May-Jun), Varsha ritu (July-Aug), Sharad ritu (Sep-Oct), and Hemant ritu (Nov–Dec). The source of collected plant material was authenticated at Central Ayurveda Research Institute (CARI), Jhansi. The voucher specimen was deposited in the CARI herbarium (Accession no. 23602) for future reference. Botanical identification and comparative macro and microscopic studies for samples collected in each season were carried out. The extraction in various solvents such as alcohol, hydro alcohol, and methanol through soxhlet was carried out to evaluate the quantitative analysis of extractive values and asiaticoside in all six seasons through HPLC at Captain Srinivasa Murthy Central Ayurveda Research Institute (CSMCARI), Chennai. RESULTS: The whole plant is used for medicinal purpose. It was observed that the flowering and fruiting occur at end of the rainy (Varsha ritu) season and persists up to the winter season (Shishir ritu). Plant material is procured from the same habitat in all seasons from Central Ayurveda Research Institute (CARI) garden, Jhansi. The raw botanical material showed the similar characters in powder microscopy in Shishir ritu, Vasant ritu, Sharad ritu, and Hemant ritu whereas the powder study of Grishma ritu and Varsha ritu was observed without the fruiting parts that is pericarp and endosperm. And other four seasons are same as Shishir ritu. Selected marker compound asiaticoside was quantified in all available seasons by HPLC. The extractive value was found maximum of 2.2332 g (44.66% w/w) in a methanol extract of Grishma ritu sample and HPLC estimation evident for the large quantity of asiaticoside is more upto 0.4596 (% w/w) in Grishma ritu sample. CONCLUSION: The highest concentration of asiaticoside was found in the sample collected during Grishma ritu (May and June). Hence, it can be concluded that the best procurement time for Mandukaparni whole plant is Grishma ritu (May and June).
Keywords: Asiaticoside, authentication and pharmacognosy, HPLC, Mandookparni, procurement time, seasonal variation
|How to cite this article:|
Narasimhaji CV, Mangal AK, Rekha P, Ilavarasan R, Srikanth N. Seasonal assessment of asiaticoside content in Centella asiatica (L.) Urb. by HPLC and pharmacognostical studies to establish its best procurement time. J Drug Res Ayurvedic Sci 2023;8:134-42
|How to cite this URL:|
Narasimhaji CV, Mangal AK, Rekha P, Ilavarasan R, Srikanth N. Seasonal assessment of asiaticoside content in Centella asiatica (L.) Urb. by HPLC and pharmacognostical studies to establish its best procurement time. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Jun 10];8:134-42. Available from: http://www.jdrasccras.com/text.asp?2023/8/2/134/373020
| Introduction|| |
In the Ayurvedic Formulary of India, it is mentioned that Centella asiatica has to be taken wherever Manduki and Mandukaparni are mentioned., Juice of Mandukaparni is indicated for intellect promotion. In reference of juice preparation, it has been clearly mentioned by Sharangadhara that always fresh plants should be used for juice. Therefore, fresh Mandukaparni should be collected for juice preparation. The drug is reported to possess the following properties according to Ayurveda Kaphapittahara (pacify Kapha and Pitta dosha), Hrdya (cardiotonic), Medhya (nootropics), Svarya (improves voice tone and quality), Rasayana (rejuvenating agent), Dipana (carminative), Varnya (improves texture of skin), Vishaghna, Ayushya (increases longevity), Balya (tonic), Smritiprada (memory booster), Raktapittashamaka (pacify rakta and pitta dosha), Vayasthapan (rejuvenation for healthy aging), Stanyajanana (galactogogue), Stanyashodhana (breast milk purifier), Vranashodhaka (wound cleansing agents), Vranaropaka (wound healing), and Amapachana (digestive). The great surgeon of Indian Medicine Maharshi Sushrut has described it as the best shaka with Jivanti.,Mandukaparni is used in the treatment of the following therapeutic conditions raktapitta (bleeding disorders); kustha (diseases of skin); meha (excessive flow of urine); jvara (fever); svasa (asthma); kasa (cough); aruci (tastelessness); pandu (anemia); sotha (inflammation); kandu (itching); raktadosa (disorders of blood).
The collection of specific part of the plant in a particular season may possess more active principles according to Ayurveda. Example collection of leaves, branches in rainy and spring (in early summer) seasons is the best for its potency as the leaves contain maximum secondary metabolites to possess the most desirable therapeutic action. It is a small perennial trailing and slightly aromatic, creeping stoloniferous herb with slender slightly succulent stems, orbicular-crenate leaf on long petioles, and emerging at nodes, underside of which gives out fibrous roots. Inflorescence in single umbel with small bracts bearing 1–5 sessile, white, or reddish flowers.
Previous literature review suggests that plant is collected in the month of August and September, cleaned of adhering soil, and spread on polythene sheets for quick drying in shade. Economically important part of C. asiatica (L.) Urb. is the whole plant. Asiaticoside, madecassoside, and a number of tri-terpinoid saponin glycosides are active constituents., In the present study asiaticoside is taken as selected marker compound. Asiaticoside was quantified by HPLC in all six seasons procured from the same habitat. The powder, macro, and microscopic variations were recorded for all seasons. The plant shows different morphological characters as well as a difference in the content of secondary bioactive metabolite in different seasons. Hence, in the present study, an attempt was made to select an appropriate season to collect the useful part of the plant containing the maximum amount of bioactive secondary metabolite.
| Materials and Methods|| |
Collection and authentication
The whole plant of Mandukaparni (Centella asiatica) was collected from the CARI garden and authenticated at CARI, Jhansi. The collected plant sample was washed under running fresh water and subjected to shade drying for about 4 weeks. The shade-dried materials were further crushed to powder and the powder was passed through mesh number 85 and stored in an airtight container. The plant material collected in each season was verified for parameters such as physicochemical, macro and microscopic. It was found in compliance with the Ayurvedic Pharmacopeia of India.
Macroscopic and microscopic analysis
Macroscopic features were studied and photographs were taken with a Nikon DSLR camera in each season. Macroscopic characters were studied with the help of floras. A comparison of macroscopic characters for all samples collected in all seasons has been conducted. Collected plants were observed carefully for their morphological characters such as color, shape, and size. Characters of plant parts were studied as per taxonomy and measurement was taken by scale through the naked eye. Macroscopic characters & powder microscopic features in different seasons, organoleptic characters were briefed in [Table 1] and [Table 2] respectively. Microscopic examinations were carried out as per the standard method.,,,,, Transverse sections of plant parts were prepared and stained with safranin and fast green as per the procedure. The pictures were documented by using a microscope attached to the digital camera. The same procedure was followed for powder microscopy analysis.
HPLC methodology/quantitative analysis
Extraction and preparation of test solution
Each 5 g of dried powdered whole plant material was extracted with 200 ml of methanol, ethanol, and 95% of hydro alcohol by using soxhlet for 24 h and they were dried under reduced pressure. The obtained residue weights for the above extractions are given in [Table 3]. The above-obtained residues from all three extracts of each six seasons were weighed in triplicate and dissolved in methanol using a 2 ml volumetric flask, filtered through 0.22 μ membrane filter, and used for HPLC analysis.
Chemicals and reference standards
Standard asiaticoside marker was purchased from Natural Remedies Pvt. Ltd. All HPLC grade ethanol, methanol, acetonitrile, water, potassium dihydrogen orthophosphate, and orthophosphoric acid were purchased from Merck.
Standard solution: 1.8 mg of asiaticoside was dissolved in HPLC grade methanol in a 10 ml volumetric flask and volume was made up.
Buffer solution: Phosphate buffer was prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water and adding 0. 5 ml of orthophosphoric acid and making up the volume to 1000 ml.
Instrumentation and HPLC analysis
HPLC analysis was performed on Agilent Technologies 1200 series, it consists of an 1100/1200 Quaternary Pump, Manual injector, and 1200 diode array detector at 210 nm. Separation was achieved on Eclipse XBD C18 –column (dimensions 4.6 mm × 150 mm). Chromatograms were recorded by using Agilent chemstation software. The mobile phase consisted of acetonitrile: buffer (25: 75), pumped at a flow rate of 1.8 ml/min. The detection was carried out at 210 nm. The injection volume of both the standard and sample was 20 μl. The temperature in the laboratory was 25 ± 2°C. All the solutions used for the analysis were filtered through a 0.45 μm membrane filter and extracts were filtered through a 0.22 μm membrane filter using Thermo fisher and Millipore filtration unit.
Calibration and estimation of asiaticoside
The above standard was appropriately diluted further to get a concentration of 0.18, 0.09, and 0.045 mg/ml of asiaticoside. The standards were injected through a manual injector and recorded the peak areas respectively at 210 nm through a diode array detector. The calibration curve was established for peak area vs. concentration of the applied standards. 20 μl of each test solution was injected through a manual injector into the HPLC system. It was, determined the peak area of the test solution corresponding to that of asiaticoside from above calibration curve. The amount of asiaticoside content for all the available seasons of C. asiatica from various solvents was given in the [Table 4].
| Result and Discussion|| |
Salient macro and microscopic features
Macroscopic features: The dried herb consists of an interwoven mass of crumpled leaves with long wiry petioles, broken stems, and very few small flowers and fruits. The odor was strong, faintly aromatic; tastes pungent. Root—cylindrical, fibrous, tapering towards the end, somewhat tortuous, 1–3 cm in length; 0.3–0.5 cm in diameter, arising in clusters from the nodes; fracture brittle and short; light brown in color. Stem (runner)—cylindrical, with slender 5–15 cm long internodes, 1–2 mm in diameter, hollow, faintly longitudinally striated, nodes swollen, rooting at places. Leaf—reniform or orbicular, frequently cup-shaped, 3–3.5 cm in length, 2–3 cm in width, arises in clusters of 4–5 from nodes, margin crenate to lobulate, surface glabrous, upper greenish brown, lower grayish green, palmately veined; stipule broad, ovate, crenate, adnate to the petiole forming a sheathing base; petiole wiry, grooved crowded at the nodes, 5–12 cm in length [Figure 1][Figure 2][Figure 3][Figure 4][Figure 5][Figure 6].
|Figure 1: Mandookparni in fruiting in Shishir ritu, Vasant ritu, Sharad ritu, and Hemant ritu|
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|Figure 5: Transverse section of C. asiatica leaf passing through the midrib. (A), 4× (B),10×. col, collenchymas; le, lower epidermis; pal, palisade; ph, phloem; rcr, rosette shaped calcium oxalate crystals; ue, upper epidermis; vb, vascular bundle; xyv, xylem vessel.|
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|Figure 6: TS of petiole of C. asiatica (C) 4×, (D) 10× acr, acicular crystals of calcium oxalate; col, collenchyma; pa, parenchyma; pi, pith; rcr, rosette shaped calcium oxalate crystals; vb, vascular bundles|
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Leaf-Transverse section (TS): TS of the leaf passing through the midrib is convex on the lower side and slightly depressed on the upper side, shows collenchymatous tissue underneath both the epidermis of the midrib, a centrally located meristele with a dorsiventral lamina and lateral extension on its both sides. Detailed TS shows a layer of upper and lower epidermis embedded with stomata, they being more on the lower side, the cells of the upper being bigger in size than that of the lower one; covered with striated cuticle, uni- to multicellular, simple, and glandular trichomes with unicellular stalk and cylindrical unicellular head are occasionally present; centrally located meristele shows radially arranged rows of vessels and an arc of phloem, 3–5 rows of collenchymatous tissue lies underneath both the epidermii; lamina shows a row of palisade underneath the upper epidermis, the remaining tissue of the mesophyll being occupied by 5–6 row of spongy parenchyma embedded with rosette and cluster crystals of calcium oxalate and obliquely cut vascular bundles.
Petiole-TS of the petiole is reniform in outline and shows a centrally located wide hollow pith encircled by a ring of vascular bundles identical to that of the stem, peripheral discontinuous band of hypodermal collenchymatous tissue surrounded by epidermis with few trichomes identical to that of the lamina, a discontinuous narrow band of collenchymatous hypodermis slander below the epidermis.
Powder microscopy. [Figure 7] and [Figure 8] show the upper and lower epidermis of the lamina in surface view with straight anticlinal walls embedded with anisocytic and anomocytic stomata, the cells of the lower epidermis are smaller in size and with striated cuticle; cluster and rosette crystals of calcium oxalate scattered as such throughout or embedded in parenchymatous cells, very few simple and glandular trichomes from petiole as described in the text, transversely cut fragments of lamina showing a layer of palisade underneath the upper epidermis, longitudinally cut fragments of annular and pitted vessels and thin-walled fibers; fragments of cork from the root in surface view; parquetry layer and sclereids from the pericarp, endosperm cells filled with aleurone grains and fixed oil globules, small sized, simple and compound starch grains scattered as such from the root. Stomata: upper and lower epidermis of the lamina in surface view with straight anticlinal walls embedded with anisocytic and anomocytic stomata. Crystals: They are cluster and rosette and are calcium oxalate crystals scattered throughout or embedded in parenchymatous cells. Trichomes: very few simple and glandular trichomes from the petiole. Vessel: longitudinally cut fragments of annular and pitted vessels and thin-walled fibers. Parts of fruit: Sclereids from the pericarp, endosperm cells filled with aleurone grains seen in powder. Cork: fragments of cork from the root in surface view. Starch grains: simple and compound starch grains scattered as such from the root.
|Figure 7: Powder microscopy of C. asiatica whole plant. (A) transversely cut cork; (B) cork in surface view; (C) fragment of fibres; (D) sclereids from fruit pericarp; (E) simple and compound starch grains; (F) pitted and spiral vessels; (G) transversely cut lamina showing palisade underneath the upper epidermis; (H) rosette crystals of calcium oxalate; (I) oil drops; (J) simple and multicellular trichomes; (K), epidermal cells of the midrib with striated cuticle; (L), lower epidermis showing anisocytic and anomocytic stomata and striated cuticle; (M), fragment of upper epidermis with stomata and underlying palisade; (N), fragment of cotyledon and endosperm; (O) cells filled with oil globules and starch grains|
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|Figure 8: Powder microscopy of C. whole plant. (A) rosette crystals of calcium oxalate; (B) fragment of fibers; (C) starch grains; (D) trichome; (E) cell filled with oil globules and starch grains; (F) pitted vessel|
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| Discussion|| |
C. asiatica (L.) Urban, is used as a brain tonic, diuretic, emmenagogue, narcotic, spasmolytic, and stimulant. It is a curative in several diseases viz., genitourinary tract, cutaneous system, urinary and ovarian irritation, itching, headache, giddiness, coma, skin tuberculosis, affections of cellular tissues, bruises, bowel complaints, inflamed and swollen parts dysentery, mental weakness and eruptions due to heat, and so on. It is an internal and external remedy to various skin diseases.,,,, It accumulates with plenty of phytochemicals, not less than 2% triterpene ester glycosides, triterpenoid saponins known as centelloids, including asiaticoside [Figure 9], centelloside, madacassoside, brahmoside, brahminoside, thankuniside, sceffoleside, centellose, asiaticacid, brahmic acid, centellic acid, madecassic acids isobrahmic acid, isothankuside, thankunic acid, 3-glucosyl quercetin, 3-glucosylkaempferol, 7-glucosylkaempferol, and polyacetylenes.,,
The present investigation was therefore undertaken to evaluate the requisite pharmacognostical standards by studying macroscopic characters [Figure 1][Figure 2][Figure 3][Figure 4], microscopic characters [Figure 5] and [Figure 6], and physicochemical parameters for the plant material Mandukaparni in compliance with Ayurvedic Pharmacopeia of India. It also made observations for the variations in the samples collected in all available seasons to document the internal structures of plants and powder [Figure 7] and [Figure 8]. It was noted that in the autumn, winter, and spring seasons it is in the flowering and fruiting stage therefore in these seasons fruit parts can be seen whereas in summer (Grishma ritu) and rainy (Varsha ritu) season fruit parts are absent in microscopy. The whole plant is used for medicinal purpose. At the end of the rainy (Varsha ritu) season, flowering and fruiting were seen and persisted up to the winter season (Shishir ritu). The plant material was procured from the same habitat (CARI garden) in all seasons were observed for powder microscopy. It was found the same characters in seasons of Shishir ritu, Vasant ritu, Sharad ritu and Hemant ritu while in the seasons of Grishm ritu and Varsha ritu is devoid of fruiting parts i.e. pericarp and endosperm (Table 1).
Asiaticoside was one of the major components of the plant, in the WHO monograph of Herba Centella and it was reported in a range of 0.776–0.804% in the Indian council for medical research monograph of quality standards on Indian medicinal plants. In the present study, extraction was carried out for whole plant material by soxhlet extraction and the residues weight of extractive value was found to be a maximum that is 2.2332 g (44.66%) in Grishma ritu sample of methanol extract [Table 3]. It was quantified in each season through HPLC and the calibration curve was established for peak area against the concentration of the applied standards with the linearity of 0.999 correlation [Figure 10] and [Figure 11]. An assay of asiaticoside was analyzed in all three optimized solvents of methanol, ethanol, and 95% hydro alcohol and found in 0.3662–0.4596% [Table 4]. These observed results of HPLC analysis with the maximum loads of asiaticoside were in the Grishma ritu sample [Table 4]. With these observations, it is to conclude that the best collection time of the C. asiatica whole plant is in Grishma ritu/summer season (May and June).
|Figure 10: HPLC Chromatogram of C. asiatica whole plant. (A) Test solution; (B) Asiaticoside standard|
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| Conclusion|| |
Mandukaparni possesses the maximum extractive values and one of its bioactive secondary metabolite, asiaticoside with a large quantity in Grishma ritu. Therefore it can be recommended that the best procurement time for the whole plant of Centella asiatica (L.) is in Grishma ritu (May and June).
Financial support and sponsorship
IMR Project funded by CCRAS, Ministry of Ayush, Government of India, New Delhi.
Conflicts of interest
There is no conflict of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3], [Table 4]